Vaccine

ABSTRACT

The disclosure relates to polypeptides and pharmaceutical compositions comprising polypeptides that find use in the prevention or treatment of cancer, in particular breast cancer, ovarian cancer and colorectal cancer. The disclosure also relates to methods of inducing a cytotoxic T cell response in a subject or treating cancer by administering pharmaceutical compositions comprising the peptides, and companion diagnostic methods of identifying subjects for treatment. The peptides comprise T cell epitopes that are immunogenic in a high percentage of patients.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/244,497, filed on Jan. 10, 2019, now issued as U.S. Pat. No.11,213,578 on Jan. 4, 2022, which is a continuation of U.S. applicationSer. No. 15/910,988, filed on Mar. 2, 2018, now issued as U.S. Pat. No.10,213,497 on Feb. 26, 2019, which claims the benefit of priority toEuropean Application No. 17159242.1, filed on Mar. 3, 2017, EuropeanApplication No. 17159243.9, filed on Mar. 3, 2017, and Great BritainApplication No. 1703809.2, filed on Mar. 9, 2017, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Dec. 18, 2021, isnamed “TBL_004C2_SL.txt” and is 269,433 bytes in size.

FIELD

The disclosure relates to polypeptides and vaccines that find use in theprevention or treatment of cancer, in particular most breast cancers,ovarian cancers and colorectal cancers.

BACKGROUND

Cancer is killing millions of people worldwide, because existing drugsdo not enable effective prevention or treatment. Current checkpointinhibitor immunotherapies that reactivate existing immune responses canprovide clinical benefit for a fraction of cancer patients. Currentcancer vaccines that induce new immune responses are poorly immunogenicand fail to benefit most patients.

Recent analyses of 63,220 unique tumors revealed that cancer vaccinesneed to be generated specifically for each patient because extensiveinter-individual tumor genomic heterogeneity (Hartmaier et al. GenomeMedicine 2017 9:16). Using state of art technologies it is currently notfeasible to scale HLA-specific cancer vaccines to large populations.

SUMMARY

In antigen presenting cells (APC) protein antigens are processed intopeptides. These peptides bind to human leukocyte antigen molecules(HLAs) and are presented on the cell surface as peptide-HLA complexes toT cells. Different individuals express different HLA molecules anddifferent HLA molecules present different peptides. Therefore, accordingto the state of the art, a peptide, or a fragment of a largerpolypeptide, is identified as immunogenic for a specific human subjectif it is presented by a HLA molecule that is expressed by the subject.In other words, the state of the art describes immunogenic peptides asHLA-restricted epitopes. However, HLA restricted epitopes induce T cellresponses in only a fraction of individuals who express the HLAmolecule. Peptides that activate a T cell response in one individual areinactive in others despite HLA allele matching. Therefore, it waspreviously unknown how an individual's HLA molecules present theantigen-derived epitopes that positively activate T cell responses.

As provided herein multiple HLAs expressed by an individual need topresent the same peptide in order to trigger a T cell response. Thefragments of a polypeptide antigen that are immunogenic for a specificindividual are those that can bind to multiple class I (activatecytotoxic T cells) or class II (activate helper T cells) HLAs expressedby that individual. For example, the inventors have discovered that thepresence of a T cell epitope that binds to at least three HLA type I ofa subject predicts an immune response in the subject to a polypeptide.

Based on this discovery the inventors have identified the T cellepitopes from certain breast, ovarian and/or colorectal cancerassociated-polypeptide antigens (cancer testis antigens (CTA)) that arecapable of binding to at least three class I HLA in a high proportion ofindividuals. These T cell epitopes, or fragments of the antigenscomprising the T cell epitopes, are useful for inducing specific immuneresponses against tumor cells expressing these antigens and for treatingor preventing cancer.

In a first aspect the disclosure provides a polypeptide that comprises afragment of up to 50 consecutive amino acids of

(a) a colorectal cancer-associated antigen selected from TSP50, EpCAM,SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6 and MAGE-A3,wherein the fragment comprises an amino acid sequence selected from anyone of SEQ ID NOs: 21 to 40 and 234 to 250;

(b) an ovarian cancer-associated antigen selected from PIWIL-4, WT1,EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME,HIWI, SURVIVIN, and AKAP-3 wherein the fragment comprises the amino acidsequence of any one of SEQ ID NOs: 272 to 301; and/or

(c) a breast cancer associated antigen selected from PIWIL-2, AKAP-4,EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME,NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1wherein the fragment comprises an amino acid sequence selected from anyone of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

In some specific cases the disclosure provides a polypeptide that

-   -   (a) is a fragment of a colorectal cancer-associated antigen        selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN,        MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein the fragment        comprises an amino acid sequence selected from any one of SEQ ID        NOs: 21 to 40 and 234 to 250; or    -   (b) comprises or consists of two or more fragments of one or        more colorectal cancer associated antigens selected from TSP50,        EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3        and LEMD1, wherein each fragment comprises a different amino        acid sequence selected from any one of SEQ ID NOs: 21 to 40 and        234 to 250, optionally wherein the fragments overlap or are        arranged end to end in the polypeptide; or    -   (c) is a fragment of a ovarian cancer-associated antigen        selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1,        SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3,        wherein the fragment comprises an amino acid sequence selected        from any one of SEQ ID NOs: 272 to 301; or    -   (d) comprises or consists of two or more fragments of one or        more ovarian cancer associated antigens selected from PIWIL-4,        WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3,        SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein each fragment        comprises a different amino acid sequence selected from any one        of SEQ ID NOs: 272 to 301, optionally wherein the fragments        overlap or are arranged end to end in the polypeptide; or    -   (e) is a fragment of a breast cancer associated antigen selected        from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN,        MAGE-A11, PRAME, MAGE-A9, HOM-TES-85, PIWIL-2, EpCAM, HIWI,        PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein the fragment        comprises the amino acid sequence from any one of SEQ ID NOs: 1        to 20, 24 and 172 to 194; or    -   (f) comprises or consists of two or more fragments of one or        more breast cancer associated antigens selected from SPAG9,        AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME,        MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4,        SP17, RHOXF-2, wherein each fragment comprises a different amino        acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24        and 172 to 194; optionally wherein the fragments overlap or are        arranged end to end in the polypeptide and.

In some specific cases the polypeptide comprises or consists offragments of

-   -   (a) TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8,        MAGE-A6, MAGE-A3 and LEMD1;    -   (b) PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2,        PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3; and/or    -   (c) SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN,        MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI,        PLU-1, TSGA10, ODF-4, SP17, RHOXF-2;        wherein each fragment comprises a different amino acid sequence        selected from SEQ ID NOs: 21 to 40 and 234 to 250; SEQ ID NOs:        272 to 301; and/or SEQ ID NOs: 1 to 20, 24 and 172 to 194.

In some cases the polypeptide comprises or consists of one or more aminoacid sequences selected from SEQ ID NOs: 41-80, 251 to 271, 302 to 331and 196 to 233.

In some cases the polypeptide comprises or consists of one or more aminoacid sequences selected from SEQ ID NOs: 41-80, 195-233, 251-271 and302-331 or selected from SEQ ID NOs: 81-142, 332-346, and 435-449.

In a further aspect the disclosure provides a panel of two or morepolypeptides as described above, wherein each peptide comprises orconsists of a different amino acid sequence selected from SEQ ID NOs: 21to 40 and 234 to 250; or selected from SEQ ID NOs: 272 to 301; orselected from SEQ ID NOs: 1 to 20, 24 and 172 to 194; or selected fromSEQ ID NOs: 1 to 40, 234 to 250, 272 to 301 and 172 to 194. In somecases the panel of polypeptides comprises or consists of one or morepeptides comprising or consisting of the amino acid sequences of SEQ IDNOs: 130, 121, 131, 124, 134, 126 and/or SEQ ID NOs: 435-449.

In a further aspect the disclosure provides a pharmaceutical compositionor kit having one or more polypeptides or panels of peptides asdescribed above as active ingredients, or having a polypeptidecomprising at least two amino acid sequences selected from SEQ ID NOs:21 to 40 and 234 to 250; SEQ ID NOs: 272 to 301; and/or SEQ ID NOs: 1 to20, 24 and 172 to 194 as an active ingredient; or selected from SEQ IDNOs: 130, 121, 131, 124, 134, 126 and/or 435-449 as an activeingredient.

In a further aspect the disclosure provides a method of inducing immuneresponses, (e.g. vaccination, providing immunotherapy or inducing acytotoxic T cell response in a subject), the method comprisingadministering to the subject a pharmaceutical composition, kit or thepanel of polypeptides as described above. The method may be a method oftreating cancer, such as breast cancer, ovarian cancer or colorectalcancer.

In further aspects, the disclosure provides

-   -   the pharmaceutical composition, kit or panel of polypeptides        described above for use in a method of inducing immune responses        or for use in a method of treating cancer, optionally breast        cancer, ovarian cancer or colorectal cancer; and    -   use of a peptide or a panel of peptides as described above in        the manufacture of a medicament for inducing immune responses or        for treating cancer, optionally breast cancer, ovarian cancer or        colorectal cancer.

In a further aspect the disclosure provides a method of identifying ahuman subject who will likely have a cytotoxic T cell response toadministration of a pharmaceutical composition as described above, themethod comprising

-   -   (i) determining that the active ingredient polypeptide(s) of the        pharmaceutical composition comprise a sequence that is a T cell        epitope capable of binding to at least three HLA class I of the        subject; and    -   (ii) identifying the subject as likely to have a cytotoxic T        cell response to administration of the pharmaceutical        composition.

In a further aspect the disclosure provides a method of identifying asubject who will likely have a clinical response to a method oftreatment as described above, the method comprising

-   -   (i) determining that the active ingredient polypeptide(s)        comprise two or more different amino acid sequences each of        which is        -   a. a T cell epitope capable of binding to at least three HLA            class I of the subject; and        -   b. a fragment of a cancer-associated antigen expressed by            cancer cells of the subject; and    -   (ii) identifying the subject as likely to have a clinical        response to the method of treatment.

In a further aspect the disclosure provides a method of determining thelikelihood that a specific human subject will have a clinical responseto a method of treatment, wherein one or more of the following factorscorresponds to a higher likelihood of a clinical response:

-   -   (a) presence in the active ingredient polypeptide(s) of a higher        number of amino acid sequences and/or different amino acid        sequences that are each a T cell epitope capable of binding to        at least three HLA class I of the subject;    -   (b) a higher number of target polypeptide antigens, comprising        at least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; optionally wherein the target            polypeptide antigens are expressed in the subject, further            optionally wherein the target polypeptides antigens are in            one or more samples obtained from the subject;    -   (c) a higher probability that the subject expresses target        polypeptide antigens, optionally a threshold number of the        target polypeptide antigens and/or optionally target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; and/or    -   (d) a higher number of target polypeptide antigens that the        subject is predicted to express, optionally a higher number of        target polypeptide antigens that the subject expresses with a        threshold probability, and/or optionally the target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject.

In some cases the cancer-associated antigens may be TSP50, EpCAM, SPAG9,CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6, MAGE-A3, PIWIL-4, WT1,BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, PRAME, HIWI, PLU-1,TSGA10, ODF-4, RHOXF-2, NY-SAR-35, MAGE-A9, NY-BR-1, MAGE-A11,HOM-TES-85, NY-ESO-1 and AKAP-3. In some cases the methods abovecomprise the step of determining that one or more cancer-associatedantigens is expressed by cancer cells of the subject. Thecancer-associated antigen(s) may be present in one or more samplesobtained from the subject

In some cases administration of the pharmaceutical composition or theactive ingredient polypeptides of the kit may then be selected as amethod of treatment for the subject. The subject may further be treatedby administration of the pharmaceutical composition or the activeingredient polypeptides.

In a further aspect the disclosure provides a method of treatment asdescribed above, wherein the subject has been identified as likely tohave a clinical response or as having above a threshold minimumlikelihood of having a clinical response to the treatment by the methoddescribed above.

In a further aspect the disclosure provides a method of identifying ahuman subject who will likely not have a clinical response to a methodof treatment as described above, the method comprising

-   -   (i) determining that the active ingredient polypeptide(s) of the        pharmaceutical composition do not comprise two or more different        amino acid sequences each of which is a T cell epitope capable        of binding to at least three HLA class I of the subject; and    -   (ii) identifying the subject as likely not to have a clinical        response to the method of treatment.

The methods described above may comprise the step of determining the HLAclass I genotype of the subject.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more peptides, wherein each peptide comprises adifferent one of the amino acid sequence of any one of SEQ ID NOs: 112to 142. In some embodiments, the composition comprises 2 or morepeptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, or6 or more peptides. In some embodiments, the composition comprises twopeptides, wherein each peptide comprises a different one of the aminoacid sequences of SEQ ID NOs: 121 and 124. In some embodiments, thecomposition comprises four peptides, wherein each peptide comprises adifferent one of the amino acid sequences of SEQ ID NOs: 126, 130, 131,and 134. In some embodiments, the composition comprises six peptides,wherein each peptide comprises a different one of the amino acidsequences of SEQ ID NOs: 121, 124, 126, 130, 131, and 134. In someembodiments, the composition further comprises at least one additionalpeptide comprising a fragment of an antigen selected from TSP50, EpCAM,SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, and MAGE-A6. In someembodiments, the composition further comprises one or more additionalpeptides, each of the one or more additional peptides comprising adifferent one of the amino acid sequence of any one of SEQ ID NOs:112-120, 122, 123, 125, 127-129, 132, 133, and 135-142. In someembodiments, the composition further comprises a pharmaceuticallyacceptable adjuvant, diluent, carrier, preservative, or combinationthereof. In some embodiments, the the adjuvant is selected from thegroup consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole,azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpethemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant(incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin,pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheriatoxin (DT), and combinations thereof.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more nucleic acid molecules encoding one or morepeptides, wherein each peptide comprises a different one of the aminoacid sequence of any one of SEQ ID NOs: 112 to 142.

Disclosed herein in certain embodiments are methods of identifying andtreating a human subject having cancer who will likely have a clinicalresponse to administration of a pharmaceutical composition of thedisclosure, the method comprising (i) assaying a biological sample ofthe subject to determine HLA genotype of the subject; (ii) determiningthat the pharmaceutical composition comprises two or more sequences thatare a T cell epitope capable of binding to at least three HLA class Imolecules of the subject; (iii) determining the probability that a tumorof the subject expresses one or more antigen corresponding to the T cellepitopes identified in step (ii) using population expression data foreach antigen, to identify the likelihood of the subject to have aclinical response to administration of the pharmaceutical composition;and (iv) administering the composition of the disclosure to theidentified subject. In some embodiments, the subject has colorectalcancer. In some embodiments, the pharmaceutical composition comprises 2or more peptides, 3 or more peptides, 4 or more peptides, 5 or morepeptides, or 6 or more peptides. In some embodiments, the pharmaceuticalcomposition comprises two peptides, wherein each peptide comprises adifferent one of the amino acid sequences of SEQ ID NOs: 121 and 124. Insome embodiments, the pharmaceutical composition comprises fourpeptides, wherein each peptide comprises a different one of the aminoacid sequences of SEQ ID NOs: 126, 130, 131, and 134. In someembodiments, the pharmaceutical composition comprises six peptides,wherein each peptide comprises a different one of the amino acidsequences of SEQ ID NOs: 121, 124, 126, 130, 131, and 134. In someembodiments, the pharmaceutical composition further comprises at leastone additional peptide comprising a fragment of an antigen selected fromTSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, and MAGE-A6. Insome embodiments, the pharmaceutical composition further comprises oneor more additional peptides, each of the one or more additional peptidescomprising a different one of the amino acid sequence of any one of SEQID NOs: 112-120, 122, 123, 125, 127-129, 132, 133, and 135-142. In someembodiments, the pharmaceutical composition further comprises apharmaceutically acceptable adjuvant, diluent, carrier, preservative, orcombination thereof. In some embodiments, the adjuvant is selected fromthe group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole,azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpethemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant(incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin,pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheriatoxin (DT), and combinations thereof. In some embodiments, the methodfurther comprises administering a chemotherapeutic agent, a checkpointinhibitor, a targeted therapy, radiation therapy, another immunotherapy,or combination thereof to the identified subject. In some embodiments,the method further comprises prior to the administering step, (i)assaying a tumor sample from the subject to determine that the three ormore peptides of the pharmaceutical composition comprise two or moredifferent amino acid sequences each of which is a) a fragment of acancer-associated antigen expressed by cancer cells of the subject asdetermined in step (i); and b) a T cell epitope capable of binding to atleast three HLA class I molecules of the subject; and (ii) confirmingthe subject as likely to have a clinical response to the method oftreatment.

Disclosed herein in certain embodiments are kits comprising: a firstpharmaceutical composition comprising one or more peptides, wherein eachpeptide comprises a different one of the amino acid sequence of any oneof SEQ ID NOs: 112 to 142; and a second different pharmaceuticalcomposition comprising one or more peptides, wherein each peptidecomprises a different one of the amino acid sequence of any one of SEQID NOs: 112 to 142.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising: a nucleic acid molecule expressing two or more polypeptides,each polypeptide comprising a fragment of up to 50 consecutive aminoacids of an antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39,SURVIVIN, MAGE-A8, and MAGE-A6, wherein each fragment comprises adifferent amino acid sequence selected from any one of SEQ ID NOs: 21-40and 234 to 250. In some embodiments, the polypeptides do not compriseamino acid sequences that are adjacent to each other in a correspondingantigen.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more peptides, wherein each peptide comprises adifferent one of the amino acid sequence of any one of SEQ ID NOs: 81 to111 and 435 to 449. In some embodiments, the composition comprises 2 ormore peptides, 3 or more peptides, 4 or more peptides, 5 or morepeptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9or more peptides, 10 or more peptides, 11 or more peptides, or 12 ormore peptides. In some embodiments, the composition comprises 9peptides, wherein each peptide comprises a different one of the aminoacid sequences of SEQ ID NOs: 92, 93, 98, 99-101, and 103-105. In someembodiments, the composition further comprises at least one additionalpeptide comprising a fragment of an antigen selected from PIWIL-2,AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2,PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 andNY-ESO-1. In some embodiments, the fragment of an antigen comprises anamino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and172 to 194. In some embodiments, the fragment of an antigen comprises anamino acid sequence selected from any one of SEQ ID NOs:41-60 and195-233. In some embodiments, the composition further comprises apharmaceutically acceptable adjuvant, diluent, carrier, preservative, orcombination thereof. In some embodiments, the adjuvant is selected fromthe group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole,azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpethemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant(incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin,pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheriatoxin (DT), and combinations thereof.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more nucleic acid molecules encoding one or morepeptides, wherein each peptide comprises a different one of the aminoacid sequence of any one of SEQ ID NOs: 81 to 111 and 435 to 449 In someembodiments, the one or more nucleic acid molecules encode 2 or morepeptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6or more peptides, 7 or more peptides, 8 or more peptides, 9 or morepeptides, 10 or more peptides, 11 or more peptides, or 12 or morepeptides. In some embodiments, the one or more nucleic acid moleculesencode 9 peptides, wherein each peptide comprises a different one of theamino acid sequences of SEQ ID NOs: 92, 93, 98, 99-101, and 103-105. Insome embodiments, the one or more nucleic acid molecules encode at leastone additional peptide comprising a fragment of an antigen selected fromPIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17,RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11,HOM-TES-85 and NY-ESO-1. In some embodiments, the fragment of an antigencomprises an amino acid sequence selected from any one of SEQ ID NOs: 1to 20, 24 and 172 to 194. In some embodiments, the fragment of anantigen comprises an amino acid sequence selected from any one of SEQ IDNOs:41-60 and 195-233. In some embodiments, the composition furthercomprises a pharmaceutically acceptable adjuvant, diluent, carrier,preservative, or combination thereof. In some embodiments, the adjuvantis selected from the group consisting of Montanide ISA-51, QS-21,GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacteriumparvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene(DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete),Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum),lysolecithin, pluronic polyols, polyanions, oil emulsions,dinitrophenol, diphtheria toxin (DT), and combinations thereof.

Disclosed herein in certain embodiments are methods of identifying andtreating a human subject having cancer who will likely have a clinicalresponse to administration of a pharmaceutical composition of thedisclosure, the method comprising (i) assaying a biological sample ofthe subject to determine HLA genotype of the subject; (ii) determiningthat the pharmaceutical composition comprises two or more sequences thatare a T cell epitope capable of binding to at least three HLA class Imolecules of the subject; (iii) determining the probability that a tumorof the subject expresses one or more antigen corresponding to the T cellepitopes identified in step (ii) using population expression data foreach antigen, to identify the likelihood of the subject to have aclinical response to administration of the pharmaceutical composition;and (iv) administering the composition of the disclosure to theidentified subject. In some embodiments, the subject has breast cancer.In some embodiments, the pharmaceutical composition comprises 2 or morepeptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6or more peptides, 7 or more peptides, 8 or more peptides, 9 or morepeptides, 10 or more peptides, 11 or more peptides, or 12 or morepeptides. In some embodiments, the pharmaceutical composition comprises9 peptides, wherein each peptide comprises a different one of the aminoacid sequences of SEQ ID NOs: 92, 93, 98, 99-101, and 103-105. In someembodiments, the pharmaceutical composition further comprises comprisingat least one additional peptide comprising a fragment of an antigenselected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10,ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN,MAGE-A11, HOM-TES-85 and NY-ESO-1. In some embodiments, the fragment ofan antigen comprises an amino acid sequence selected from any one of SEQID NOs: 1 to 20, 24 and 172 to 194. In some embodiments, the fragment ofan antigen comprises an amino acid sequence selected from any one of SEQID NOs:41-60 and 195-233. In some embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable adjuvant,diluent, carrier, preservative, or combination thereof. In someembodiments, the adjuvant is selected from the group consisting ofMontanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof. In some embodiments, the method further comprisesadministering a chemotherapeutic agent, a checkpoint inhibitor, atargeted therapy, radiation therapy, another immunotherapy, orcombination thereof to the identified subject. In some embodiments, themethod further comprises prior to the administering step, (i) assaying atumor sample from the subject to determine that the three or morepeptides of the pharmaceutical composition comprise two or moredifferent amino acid sequences each of which is a) a fragment of acancer-associated antigen expressed by cancer cells of the subject asdetermined in step (i); and b) a T cell epitope capable of binding to atleast three HLA class I molecules of the subject; and confirming thesubject as likely to have a clinical response to the method oftreatment.

Disclosed herein in certain embodiments are methods of identifying andtreating a human subject having cancer who will likely have an immuneresponse to administration of a pharmaceutical composition of thedisclosure, the method comprising (i) assaying a biological sample ofthe subject to determine HLA genotype of the subject; (ii) determiningthat the pharmaceutical composition comprises one or more sequences thatare a T cell epitope capable of binding to at least three HLA class Imolecules of the subject; and (iii) administering the composition of thedisclosure to the identified subject.

Disclosed herein in certain embodiments are kits comprising: a firstpharmaceutical composition comprising one or more peptides, wherein eachpeptide comprises a different one of the amino acid sequence of any oneof SEQ ID NOs: 81-111 and 435 to 449; and a second differentpharmaceutical composition comprising one or more peptides, wherein eachpeptide comprises a different one of the amino acid sequence of any oneof SEQ ID NOs: 81-111 and 435 to 449.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising: a nucleic acid molecule expressing two or more polypeptides,each polypeptide comprising a fragment of up to 50 consecutive aminoacids of an antigen selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI,SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9,NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1, wherein eachfragment comprises a different amino acid sequence selected from any oneof SEQ ID NOs: 1 to 20, 24, and 172 to 194. In some embodiments, thepolypeptides do not comprise amino acid sequences that are adjacent toeach other in a corresponding antigen.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more peptides, wherein each peptide comprises adifferent one of the amino acid sequence of any one of SEQ ID NOs:332-346. In some embodiments, the composition comprises 2 or morepeptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6or more peptides, 7 or more peptides, 8 or more peptides, 9 or morepeptides, 10 or more peptides, 11 or more peptides, 12 or more peptides,13 or more peptides, 14 or more peptides, or 15 or more peptides. Insome embodiments, the composition comprises 15 peptides, wherein eachpeptide comprises a different one of the amino acid sequences of SEQ IDNOs: 332-346. In some embodiments, the composition further comprises atleast one additional peptide comprising a fragment of an antigenselected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17,PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3. In someembodiments, the fragment comprises an amino acid sequence selected fromany one of SEQ ID NOs: 272-301. In some embodiments, the fragmentcomprises an amino acid sequence selected from any one of SEQ IDNOs:302-331. In some embodiments, the composition further comprises apharmaceutically acceptable adjuvant, diluent, carrier, preservative, orcombination thereof. In some embodiments, the adjuvant is selected fromthe group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole,azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpethemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant(incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin,pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheriatoxin (DT), and combinations thereof.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising one or more nucleic acid molecules encoding one or morepeptides, wherein each peptide comprises a different one of the aminoacid sequence of any one of SEQ ID NOs: 332-346. In some embodiments,the one or more nucleic acid molecules encode 2 or more peptides, 3 ormore peptides, 4 or more peptides, 5 or more peptides, 6 or morepeptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10or more peptides, 11 or more peptides, 12 or more peptides, 13 or morepeptides, 14 or more peptides, or 15 or more peptides. In someembodiments, the one or more nucleic acid molecules encode 15 peptides,wherein each peptide comprises a different one of the amino acidsequences of SEQ ID NOs: 332-346. In some embodiments, the one or morenucleic acid molecules encode at least one additional peptide comprisinga fragment of an antigen selected from PIWIL-4, WT1, EpCAM, BORIS,AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN,and AKAP-3. In some embodiments, the fragment comprises an amino acidsequence selected from any one of SEQ ID NOs: 272-301. In someembodiments, the fragment comprises an amino acid sequence selected fromany one of SEQ ID NOs:302-331. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable adjuvant, diluent,carrier, preservative, or combination thereof. In some embodiments, theadjuvant is selected from the group consisting of Montanide ISA-51,QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG),corynbacterium parvum, levamisole, azimezone, isoprinisone,dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freundsadjuvant (complete), Freunds adjuvant (incomplete), mineral gels,aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions,oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinationsthereof.

Disclosed herein in certain embodiments are methods of identifying andtreating a human subject having cancer who will likely have a clinicalresponse to administration of a pharmaceutical composition according ofthe disclosure, the method comprising (i) assaying a biological sampleof the subject to determine HLA genotype of the subject; (ii)determining that the pharmaceutical composition comprises two or moresequences that are a T cell epitope capable of binding to at least threeHLA class I molecules of the subject; (iii) determining the probabilitythat a tumor of the subject expresses one or more antigen correspondingto the T cell epitopes identified in step (ii) using populationexpression data for each antigen, to identify the likelihood of thesubject to have a clinical response to administration of thepharmaceutical composition; and (iv) administering the composition of ofthe disclosure to the identified subject. In some embodiments, thesubject has ovarian cancer. In some embodiments, the pharmaceuticalcomposition comprises 2 or more peptides, 3 or more peptides, 4 or morepeptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8or more peptides, 9 or more peptides, 10 or more peptides, 11 or morepeptides, 12 or more peptides, 13 or more peptides, 14 or more peptides,or 15 or more peptides. In some embodiments, the pharmaceuticalcomposition comprises 15 peptides, wherein each peptide comprises adifferent one of the amino acid sequences of SEQ ID NOs: 332-346. Insome embodiments, the pharmaceutical composition further comprisescomprising at least one additional peptide comprising a fragment of anantigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1,SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3. Insome embodiments, the fragment comprises an amino acid sequence selectedfrom any one of SEQ ID NOs: 272-301 In some embodiments, the fragmentcomprises an amino acid sequence selected from any one of SEQ IDNOs:302-331. In some embodiments, the pharmaceutical composition furthercomprises a pharmaceutically acceptable adjuvant, diluent, carrier,preservative, or combination thereof. In some embodiments, the adjuvantis selected from the group consisting of Montanide ISA-51, QS-21,GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacteriumparvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene(DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete),Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum),lysolecithin, pluronic polyols, polyanions, oil emulsions,dinitrophenol, diphtheria toxin (DT), and combinations thereof. In someembodiments, the method further comprises administering achemotherapeutic agent, a checkpoint inhibitor, a targeted therapy,radiation therapy, another immunotherapy, or combination thereof to theidentified subject. In some embodiments, the method further comprisesprior to the administering step, (i) assaying a tumor sample from thesubject to determine that the three or more peptides of thepharmaceutical composition comprise two or more different amino acidsequences each of which is a) a fragment of a cancer-associated antigenexpressed by cancer cells of the subject as determined in step (i); andb) a T cell epitope capable of binding to at least three HLA class Imolecules of the subject; and (ii) confirming the subject as likely tohave a clinical response to the method of treatment. Disclosed herein incertain embodiments are methods of identifying and treating a humansubject having cancer who will likely have an immune response toadministration of a pharmaceutical composition of the disclosure, themethod comprising (i) assaying a biological sample of the subject todetermine HLA genotype of the subject; (ii) determining that thepharmaceutical composition comprises one or more sequences that are a Tcell epitope capable of binding to at least three HLA class I moleculesof the subject; and (iii) administering the composition of thedisclosure to the identified subject.

Disclosed herein in certain embodiments are kits comprising: a firstpharmaceutical composition comprising one or more peptides, wherein eachpeptide comprises a different one of the amino acid sequence of any oneof SEQ ID NOs: 332-346; and a second different pharmaceuticalcomposition comprising one or more peptides, wherein each peptidecomprises a different one of the amino acid sequence of any one of SEQID NOs: 332-346.

Disclosed herein in certain embodiments are pharmaceutical compositionscomprising: a nucleic acid molecule expressing two or more polypeptides,each polypeptide comprising a fragment of up to 50 consecutive aminoacids of an antigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4,OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, andAKAP-3, wherein each fragment comprises a different amino acid sequenceselected from any one of SEQ ID NOs: 272-301. In some embodiments, thepolypeptides do not comprise amino acid sequences that are adjacent toeach other in a corresponding antigen.

Disclosure

The disclosure will now be described in more detail, by way of exampleand not limitation, and by reference to the accompanying drawings. Manyequivalent modifications and variations will be apparent, to thoseskilled in the art when given this disclosure. Accordingly, theexemplary embodiments of the disclosure set forth are considered to beillustrative and not limiting. Various changes to the describedembodiments may be made without departing from the scope of thedisclosure. All documents cited herein, whether supra or infra, areexpressly incorporated by reference in their entirety.

The present disclosure includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. As used in thisspecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a peptide” includes two ormore such peptides.

Section headings are used herein for convenience only and are not to beconstrued as limiting in any way.

DESCRIPTION OF THE FIGURES

FIG. 1 —ROC curve of HLA restricted PEPI biomarkers.

FIG. 2 —ROC curve of ≥1 PEPI3+ Test for the determination of thediagnostic accuracy.

FIGS. 3A-B—Distribution of HLA class I PEPI3+ compared to CD8+ T cellresponses measured by a state of art assay among peptide pools used inthe CD8+ T cell response assays. FIG. 3A: HLA class I restrictedPEPI3+s. The 90% Overall Percent of Agreement (OPA) among the T cellresponses and PEPI3+ peptides demonstrate the utility of the inventedpeptides for prediction of vaccine induced T cell response set ofindividuals. FIG. 3B: Class I HLA restricted epitopes (PEPI1+). The OPAbetween predicted epitopes and CD8+ T cell responses was 28% (notstatistically significant). Darkest grey: True positive (TP), bothpeptide and T cell responses were detected; Light grey: False negative(FN), only T cell responses were detected; Lightest grey: False positive(FP), only peptide were detected; Dark grey: True negative (TN): neitherpeptides nor T cell responses were detected.

FIGS. 4A-B—Distribution of HLA class II PEPIs compared to CD4+ T cellresponses measured by a state of art assay among peptide pools used inthe assays. FIG. 4A: HLA class II restricted PEPI4+s. 67% OPA betweenPEPI4+ and CD4+ T-cell responses (p=0.002). FIG. 4B: The class II HLArestricted epitopes. OPA between class II HLA restricted epitopes andCD4+ T cell responses was 66% (not statistically significant). Darkestgrey: True positive (TP), both peptide and T cell responses weredetected; Light grey: False negative (FN), only T cell responses weredetected; Lightest grey: False positive (FP), only peptide weredetected; Dark grey: True negative (TN): neither peptides nor T cellresponses were detected.

FIGS. 5A-D—Multiple HLA binding peptides that define the HPV-16 LPVvaccine specific T cell response set of 18 VIN-3 and 5 cervical cancerpatients. HLA class I restricted PEPI3 counts (FIGS. 5A and 5B) and HLAclass II restricted PEPI3 counts (FIGS. 5C and 5C) derived from LPVantigens of each patient. Light grey: immune responders measured aftervaccination in the clinical trial; Dark grey: Immune non-respondersmeasured after vaccination in the clinical trial. Results show that ≥3HLA class I binding peptides predict the CD8+ T cell reactivity and ≥4HLA class II binding peptides predict the CD4+ T cell reactivity.

FIG. 6 —The multiple HLA class I binding peptides that define the HPVvaccine specific T cell response set of 2 patients. Panel A: Four HPVantigens in the HPV vaccine. Boxes represent the length of the aminoacid sequences from the N terminus to the C terminus. Panel B: Processto identify the multiple HLA binding peptides of two patients: HLAsequences of the patients labelled as 4-digit HLA genotype right fromthe patient's ID. The location of the 1^(st) amino acid of the 54 and 91epitopes that can bind to the patient 12-11 and patient 14-5 HLAs(PEPI1+) respectively are depicted with lines. PEPI2 represents thepeptides selected from PEPI1+s that can bind to multiple HLAs of apatient (PEPI2+). PEPI3 represent peptides that can bind to ≥3 HLAs of apatient (PEPI3+). PEPI4 represent peptides that can bind to ≥4 HLAs of apatient (PEPI4+). PEPI5 represent peptides that can bind to ≥5 HLAs of apatient (PEPI5+). PEPI6 represent peptides that can bind to 6 HLAs of apatient (PEPI6). Panel C: The DNA vaccine specific PEPI3+ set of twopatients characterizes their vaccine specific T cell responses.

FIG. 7 —Correlation between the ≥1 PEPI3+ Score and CTL response ratesof peptide targets determined in clinical trials.

FIG. 8 —Correlation between the ≥1 PEPI3+ Score and the clinical ImmuneResponse Rate (IRR) of immunotherapy vaccines. Dashed lines: 95%confidence band.

FIG. 9 —Correlation between the ≥2 PEPI3+ Score and Disease Control Rate(DCR) of immunotherapy vaccines. Dashed lines: 95% confidence band.

FIG. 10 —Peptide hotspot analysis example: PRAME antigen hotspot on 433patients of the Model Population. On the y axis are the 433 patients ofthe Model Population, on the x axis is the amino acid sequence of thePRAME antigen (CTA). Each data point represents a PEPI presented by 3HLA class I of one patient starting at the specified amino acidposition. The two most frequent PEPIs (called bestEPIs) of the PRAMEantigen are highlighted in dark gray (peptide hotspots=PEPI Hotspots).

FIG. 11 —CTA Expression Curve calculated by analyzing expressionfrequency data of tumor specific antigens (CTAs) in human breast cancertissues. (No cell line data were included.)

FIGS. 12A-B—Antigen expression distribution for breast cancer based onthe calculation of multi-antigen responses from expression frequenciesof the selected 10 different CTAs. FIG. 12A: non-cumulative distributionto calculate the expected value for the number of expressed antigens(AG50). This value shows that probably 6.14 vaccine antigens will beexpressed by breast tumor cells. FIG. 12B: cumulative distribution curveof the minimum number of expressed antigens (CTA expression curve). Thisshows that minimum 4 vaccine antigens will be expressed with 95%probability in breast cancer cell (AG95).

FIGS. 13A-B—PEPI representing antigens: breast cancer vaccine-specificCTA antigens with ≥1 PEPI, called as “AP”) distribution within the ModelPopulation (n=433) for breast cancer vaccine. FIG. 13A: non-cumulativedistribution of AP where the average number of APs is: AP50=5.30,meaning that in average almost 6 CTAs will have PEPIs in the ModelPopulation. FIG. 13B: cumulative distribution curve of the minimumnumber of APs in the Model Population (n=433). This shows that at leastone vaccine antigen will have PEPIs in 95% of the Model Population(n=433) (AP95=1).

FIGS. 14A-B—PEPI represented expressed antigen (breast cancervaccine-specific CTA antigens expressed by the tumor, for which ≥1 PEPIis predicted, called as “AGP”) distribution within the model population(n=433) calculated with CTA expression rates for breast cancer. FIG.14A: non-cumulative distribution of AGP where the expected value fornumber expressed CTAs represented by PEPI is AGP50=3.37. AGP50 is ameasure of the effectiveness of the disclosed breast cancer vaccine inattacking breast tumor in an unselected patient population. AGP50=3.37means that at least 3 CTAs from the vaccine will probably be expressedby the breast tumor cells and present PEPIs in the Model Population.FIG. 14B: cumulative distribution curve of the minimum number of AGPs inthe Model Population (n=433) shows that at least 1 of the vaccine CTAswill present PEPIs in 92% of the population and the remaining 8% of thepopulation will likely have no AGP at all (AGP95=0, AGP92=1).

FIG. 15 —CTA Expression Curve calculated by analyzing expressionfrequency data of tumor specific antigens (CTAs) in human colorectalcancer tissues. (No cell line data were included.)

FIGS. 16A-B—Antigen expression distribution for colorectal cancer basedon the calculation of multi-antigen responses from expressionfrequencies of the selected 7 different CTAs. FIG. 16A: non-cumulativedistribution to calculate the expected vale for the number of expressedvaccine antigens in colorectal cancers (AG50). This value shows thatprobably 4.96 vaccine antigens will be expressed by colorectal tumorcells. FIG. 16B: cumulative distribution curve of the minimum number ofexpressed antigens (CTA expression curve). This shows that minimum 3antigens will be expressed with 95% probability in the colorectal cancercell (AG95).

FIGS. 17A-B—PEPI represented antigen (colorectal cancer vaccine-specificCTA antigens for which ≥1 PEPI is predicted. Called as “AP”)distribution within the model population (n=433) for colorectal cancer.FIG. 17A: non-cumulative distribution of AP where the average number ofAPs is: AP50=4.73, meaning that in average 5 CTAs will be represented byPEPIs in the model population FIG. 17B: cumulative distribution curve ofthe minimum number of APs in the model population (n=433). This showsthat 2 or more antigens will be represented by PEPIs in 95% of the modelpopulation (n=433) (AP95=2).

FIGS. 18A-B—PEPI represented expressed antigen (colorectal cancervaccine-specific CTA antigens expressed by the tumor, for which ≥1 PEPIis predicted. Called as “AGP”) distribution within the model population(n=433) calculated with CTA expression rates for colorectal cancer. FIG.18A: non-cumulative distribution of AGP where the expected value fornumber expressed CTAs represented by PEPI is AGP50=2.54. AGP50 is ameasure of the effectiveness of the disclosed colorectal cancer vaccinein attacking colorectal tumors in an unselected patient population.AGP50=2.54 means that at least 2-3 CTAs from the vaccine will probablybe expressed by the colorectal tumor cells and present PEPIs in theModel Population. FIG. 18B: cumulative distribution curve of the minimumnumber of AGPs in the Model Population (n=433) shows that at least 1 ofthe vaccine CTAs will be expressed and also present PEPIs in 93% of thepopulation (AGP93=1).

FIG. 19 —Schematic showing exemplary positions of amino acids inoverlapping HLA class I- and HLA class-II binding epitopes in a 30-merpeptide.

FIGS. 20A-B—Antigenicity of PolyPEPI1018 CRC Vaccine in a generalpopulation. The antigenicity of PolyPEPI1018 in a subject is determinedby the AP count, which indicates the number of vaccine antigens thatinduce T cell responses in a subject. The AP count of PolyPEPI1018 wasdetermined in each of the 433 subjects in the Model Population using thePEPI Test, and the AP50 count was then calculated for the ModelPopulation. The AP50 of PolyPEPI1018 in the Model Population is 4.73.The mean number of immunogenic antigens (i.e., antigens with ≥1 PEPI) inPolyPEPI1018 in a general population is 4.73. Abbreviations: AP=antigenswith ≥1 PEPI. Left Panel: Cumulative distribution curve. Right Panel:Distinct distribution curve.

FIGS. 21A-B—Effectiveness of PolyPEPI1018 CRC Vaccine in a generalpopulation. Vaccine induced T cells can recognize and kill tumor cellsif a PEPI in the vaccine is presented by the tumor cell. The number ofAGPs (expressed antigens with PEPI) is an indicator of vaccineeffectiveness in an individual, and is dependent on both the potency andantigenicity of PolyPEPI1018. The mean number of immunogenic CTAs (i.e.,AP [expressed antigens with ≥1 PEPI]) in PolyPEPI1018 is 2.54 in theModel Population. The likelihood that PolyPEPI1018 induces T cellresponses against multiple antigens in a subject (i.e., mAGP) in theModel Population is 77%.

FIGS. 22A-B—Probability of vaccine antigen expression in the XYZpatient's tumor cells. There is over 95% probability that 5 out of the12 target antigens in the vaccine regimen is expressed in the patient'stumor. Consequently, the 12 peptide vaccines together can induce immuneresponses against at least 5 ovarian cancer antigens with 95%probability (AGP95). It has 84% probability that each peptide willinduce immune responses in the XYZ patient. AGP50 is the mean (expectedvalue)=7.9 (it is a measure of the effectiveness of the vaccine inattacking the tumor of XYZ patient).

FIG. 23 —Mill findings of patient XYZ treated with personalised (PIT)vaccine. This late stage, heavily pretreated ovarian cancer patient hadan unexpected objective response after the PIT vaccine treatment. TheseMill findings suggest that PIT vaccine in combination with chemotherapysignificantly reduced her tumor burden. The patient now continues thePIT vaccine treatment.

FIGS. 24A-B—Probability of vaccine antigen expression in the ABCpatient's tumor cells. There is over 95% probability that 4 out of the13 target antigens in the vaccine is expressed in the patient's tumor.Consequently, the 12 peptide vaccines together can induce immuneresponses against at least 4 breast cancer antigens with 95% probability(AGP95). It has 84% probability that each peptide will induce immuneresponses in the ABC patient. AGP50 is the mean (expected value) of thediscrete probability distribution=6.45 (it is a measure of theeffectiveness of the vaccine in attacking the tumor of ABC patient).

DESCRIPTION OF THE SEQUENCES

SEQ ID NOs: 1 to 20 set forth 9 mer T cell epitopes described in Table17.

SEQ ID NOs: 21 to 40 set forth 9 mer T cell epitopes described in Table20.

SEQ ID NOs 41 to 60 set forth 15 mer T cell epitopes described in Table17.

SEQ ID NOs 61 to 80 set forth 15 mer T cell epitopes described in Table20.

SEQ ID NOs: 81 to 111 set forth breast cancer vaccine peptides describedin Table 18a.

SEQ ID NOs 112 to 142 set forth the colorectal cancer vaccine peptidesdescribed in Table 21a.

SEQ ID NOs 143 to 158 set forth breast cancer, colorectal cancer and/orovarian cancer associated antigens.

SEQ ID NOs 159 to 171 set forth the additional peptide sequencesdescribed in Table 10.

SEQ ID NOs 172 to 194 set forth further 9 mer T cell epitopes describedin Table 17.

SEQ ID NOs 195 to 233 set forth further 15 mer T cell epitopes describedin Table 17.

SEQ ID NOs 234 to 250 set forth further 9 mer T cell epitopes describedin Table 20.

SEQ ID NOs 251 to 271 set forth further 15 mer T cell epitopes describedin Table 20.

SEQ ID NOs: 272 to 301 set forth the 9 mer T cell epitopes described inTable 23.

SEQ ID NOs: 302 to 331 set forth the 15 mer T cell epitopes described inTable 23.

SEQ ID NOs: 332 to 346 set forth the ovarian cancer vaccine peptides setforth in Table 24.

SEQ ID NOs: 347 to 361 set forth further breast cancer, colorectalcancer and/or ovarian cancer associated antigens.

SEQ ID NOs: 362 to 374 set forth personalised vaccine peptides designedfor patient XYZ described in Table 38.

SEQ ID NOs: 375 to 386 set forth personalised vaccine peptides designedfor patient ABC described in Table 41.

SEQ ID NOs 387 to 434 set forth further 9 mer T cell epitopes describedin Table 32

SEQ ID NOs: 435 to 449 set forth further breast cancer vaccine peptidesdescribed in Table 18a.

DETAILED DESCRIPTION

HLA Genotypes

HLAs are encoded by the most polymorphic genes of the human genome. Eachperson has a maternal and a paternal allele for the three HLA class Imolecules (HLA-A*, HLA-B*, HLA-C*) and four HLA class II molecules(HLA-DP*, HLA-DQ*, HLA-DRB1*, HLA-DRB3*/4*/5*). Practically, each personexpresses a different combination of 6 HLA class I and 8 HLA class IImolecules that present different epitopes from the same protein antigen.The function of HLA molecules is to regulate T cell responses. Howeverup to date it was unknown how the HLAs of a person regulate T cellactivation.

The nomenclature used to designate the amino acid sequence of the HLAmolecule is as follows: gene name*allele:protein number, which, forinstance, can look like: HLA-A*02:25. In this example, “02” refers tothe allele. In most instances, alleles are defined by serotypes—meaningthat the proteins of a given allele will not react with each other inserological assays. Protein numbers (“25” in the example above) areassigned consecutively as the protein is discovered. A new proteinnumber is assigned for any protein with a different amino acid sequence(e.g. even a one amino acid change in sequence is considered a differentprotein number). Further information on the nucleic acid sequence of agiven locus may be appended to the HLA nomenclature, but suchinformation is not required for the methods described herein.

The HLA class I genotype or HLA class II genotype of an individual mayrefer to the actual amino acid sequence of each class I or class II HLAof an individual, or may refer to the nomenclature, as described above,that designates, minimally, the allele and protein number of each HLAgene. An HLA genotype may be determined using any suitable method. Forexample, the sequence may be determined via sequencing the HLA gene lociusing methods and protocols known in the art. Alternatively, the HLA setof an individual may be stored in a database and accessed using methodsknown in the art.

Some subjects may have two HLA alleles that encode the same HLA molecule(for example, two copies for HLA-A*02:25 in case of homozygosity). TheHLA molecules encoded by these alleles bind all of the same T cellepitopes. For the purposes of this disclosure “binding to at least twoHLA molecules of the subject” as used herein includes binding to the HLAmolecules encoded by two identical HLA alleles in a single subject. Inother words, “binding to at least two HLA molecules of the subject” andthe like could otherwise be expressed as “binding to the HLA moleculesencoded by at least two HLA alleles of the subject”.

Polyeptides

The disclosure relates to polypeptides that are derived from CTAs andthat are immunogenic for a high proportion of the human population.

As used herein, the term “polypeptide” refers to a full-length protein,a portion of a protein, or a peptide characterized as a string of aminoacids. As used herein, the term “peptide” refers to a short polypeptidecomprising between 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10,or 11, or 12, or 13, or 14, or 15 and 10, or 11, or 12, or 13, or 14, or15, or 20, or 25, or 30, or 35, or 40, or 45, or 50 or 55 or 60 aminoacids.

The terms “fragment” or “fragment of a polypeptide” as used herein referto a string of amino acids or an amino acid sequence typically ofreduced length relative to the or a reference polypeptide andcomprising, over the common portion, an amino acid sequence identical tothe reference polypeptide. Such a fragment according to the disclosuremay be, where appropriate, included in a larger polypeptide of which itis a constituent. In some cases the fragment may comprise the fulllength of the polypeptide, for example where the whole polypeptide, suchas a 9 amino acid peptide, is a single T cell epitope. In some cases thefragments referred to herein may be between 2, or 3, or 4, or 5 or 6 or7 or 8 or 9 and 20, or 25, or 30, or 35, or 40, or 45, or 50 aminoacids.

As used herein, the term “epitope” or “T cell epitope” refers to asequence of contiguous amino acids contained within a protein antigenthat possess a binding affinity for (is capable of binding to) one ormore HLAs. An epitope is HLA- and antigen-specific (HLA-epitope pairs,predicted with known methods), but not subject specific. An epitope, a Tcell epitope, a polypeptide, a fragment of a polypeptide or acomposition comprising a polypeptide or a fragment thereof is“immunogenic” for a specific human subject if it is capable of inducinga T cell response (a cytotoxic T cell response or a helper T cellresponse) in that subject. In some cases the helper T cell response is aTh1-type helper T cell response. In some cases an epitope, a T cellepitope, a polypeptide, a fragment of a polypeptide or a compositioncomprising a polypeptide or a fragment thereof is “immunogenic” for aspecific human subject if it is more likely to induce a T cell responseor immune response in the subject than a different T cell epitope (or insome cases two different T cell epitopes each) capable of binding tojust one HLA molecule of the subject.

The terms “T cell response” and “immune response” are used hereininterchangeably, and refer to the activation of T cells and/or theinduction of one or more effector functions following recognition of oneor more HLA-epitope binding pairs. In some cases an “immune response”includes an antibody response, because HLA class II molecules stimulatehelper responses that are involved in inducing both long lasting CTLresponses and antibody responses. Effector functions includecytotoxicity, cytokine production and proliferation. According to thepresent disclosure, an epitope, a T cell epitope, or a fragment of apolypeptide is immunogenic for a specific subject if it is capable ofbinding to at least two, or in some cases at least three, class I or atleast two, or in some cases at least three or at least four class IIHLAs of the subject.

For the purposes of this disclosure we have coined the term “personalepitope”, or “PEPI” to distinguish subject specific epitopes from HLAspecific epitopes. A “PEPI” is a fragment of a polypeptide consisting ofa sequence of contiguous amino acids of the polypeptide that is a T cellepitope capable of binding to one or more HLA class I molecules of aspecific human subject. In other cases a “PEPI” is a fragment of apolypeptide consisting of a sequence of contiguous amino acids of thepolypeptide that is a T cell epitope capable of binding to one or moreHLA class II molecules of a specific human subject. In other words a“PEPI” is a T cell epitope that is recognised by the HLA set of aspecific individual, and is consequently specific to the subject inaddition to the HLA and the antigen. In contrast to an “epitope”, whichis specific only to HLA and the antigen, PEPIs are specific to anindividual because different individuals have different HLA moleculeswhich each bind to different T cell epitopes. This subject specificityof the PEPIs allows to make personalized cancer vaccines.

“PEPI1” as used herein refers to a peptide, or a fragment of apolypeptide, that can bind to one HLA class I molecule (or, in specificcontexts, HLA class II molecule) of an individual. “PEPI1+” refers to apeptide, or a fragment of a polypeptide, that can bind to one or moreHLA class I molecule of an individual.

“PEPI2” refers to a peptide, or a fragment of a polypeptide, that canbind to two HLA class I (or II) molecules of an individual. “PEPI2+”refers to a peptide, or a fragment of a polypeptide, that can bind totwo or more HLA class I (or II) molecules of an individual, i.e. afragment identified according to a method of the disclosure.

“PEPI3” refers to a peptide, or a fragment of a polypeptide, that canbind to three HLA class I (or II) molecules of an individual. “PEPI3+”refers to a peptide, or a fragment of a polypeptide, that can bind tothree or more HLA class I (or II) molecules of an individual.

“PEPI4” refers to a peptide, or a fragment of a polypeptide, that canbind to four HLA class I (or II) molecules of an individual. “PEPI4+”refers to a peptide, or a fragment of a polypeptide, that can bind tofour or more HLA class I (or II) molecules of an individual.

“PEPI5” refers to a peptide, or a fragment of a polypeptide, that canbind to five HLA class I (or II) molecules of an individual. “PEPI5+”refers to a peptide, or a fragment of a polypeptide, that can bind tofive or more HLA class I (or II) molecules of an individual.

“PEPI6” refers to a peptide, or a fragment of a polypeptide, that canbind to all six HLA class I (or six HLA class II) molecules of anindividual.

Generally speaking, epitopes presented by HLA class I molecules areabout nine amino acids long and epitopes presented by HLA class IImolecules are about fifteen amino acids long. For the purposes of thisdisclosure, however, an epitope may be more or less than nine (for HLAClass I) or fifteen (for HLA Class II) amino acids long, as long as theepitope is capable of binding HLA. For example, an epitope that iscapable of binding to class I HLA may be between 7, or 8 or 9 and 9 or10 or 11 amino acids long. An epitope that is capable of binding to aclass II HLA may be between 13, or 14 or 15 and 15 or 16 or 17 aminoacids long.

A given HLA of a subject will only present to T cells a limited numberof different peptides produced by the processing of protein antigens inan APC. As used herein, “display” or “present”, when used in relation toHLA, references the binding between a peptide (epitope) and an HLA. Inthis regard, to “display” or “present” a peptide is synonymous with“binding” a peptide.

Using techniques known in the art, it is possible to determine theepitopes that will bind to a known HLA. Any suitable method may be used,provided that the same method is used to determine multiple HLA-epitopebinding pairs that are directly compared. For example, biochemicalanalysis may be used. It is also possible to use lists of epitopes knownto be bound by a given HLA. It is also possible to use predictive ormodelling software to determine which epitopes may be bound by a givenHLA. Examples are provided in Table 1. In some cases a T cell epitope iscapable of binding to a given HLA if it has an IC50 or predicted IC50 ofless than 5000 nM, less than 2000 nM, less than 1000 nM, or less than500 nM.

TABLE 1 Example software for determining epitope-HLA binding WEB ADDRESSEPITOPE PREDICTION TOOLS BIMAS, NIHwww-bimas.cit.nih.gov/molbio/hla_bind/ PPAPROC, Tubingen Univ. MHCPred,Edward Jenner Inst. of Vaccine Res. EpiJen, Edward Jenner Inst.www.ddg-pharmfac.net/epijen/EpiJen/EpiJen.htm of Vaccine Res. NetMHC,Center for Biological www.cbs.dtu.dk/services/NetMHC/ Sequence AnalysisSVMHC, Tubingen Univ. abi.inf.uni-tuebingen.de/Services/SVMHC/SYFPEITHI, Biomedicalwww.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm Informatics,Heidelberg ETK EPITOOLKIT, Tubingen Univ.etk.informatik.uni-tuebingen.de/epipred/ PREDEP, Hebrew Univ. Jerusalemmargalit.huji.ac.il/Teppred/mhc-bind/index.html RANKPEP, MIFBioinformatics bio.dfci.harvard.edu/RANKPEP/ IEDB, Immune EpitopeDatabase tools.immuneepitope.org/main/html/tcell_tools.html EPITOPEDATABASES MHCBN, Institute of Microbial www.imtech.res.in/raghava/mhcbn/Technology, Chandigarh, INDIA SYFPEITHI, Biomedical www.syfpeithi.de/Informatics, Heidelberg AntiJen, Edward Jenner Inst.www.ddg-pharmfac.net/antijen/AntiJen/antijenhomepage.htm of Vaccine Res.EPIMHC database of MHC ligands, immunax.dfci.harvard.edu/epimhc/ MIFBioinformatics IEDB, Immune Epitope Database www.iedb.org/

In some embodiments the peptides of the disclosure may comprise orconsist of one or more fragments of one or more CTAs. CTAs are nottypically expressed beyond embryonic development in healthy cells. Inhealthy adults, CTA expression is limited to male germ cells that do notexpress HLAs and cannot present antigens to T cells. Therefore, CTAs areconsidered expressional neoantigens when expressed in cancer cells.

CTAs are a good choice for cancer vaccine targets because theirexpression is (i) specific for tumor cells, (ii) more frequent inmetastases than in primary tumors and (iii) conserved among metastasesof the same patient (Gajewski ed. Targeted Therapeutics in Melanoma.Springer New York. 2012).

The peptides of the disclosure may comprise or consist of one or morefragments of one or more breast cancer associated antigens selected fromSPAG9 (SEQ ID NO: 143), AKAP-4 (SEQ ID NO: 144), BORIS (SEQ ID NO: 145),NY-SAR-35 (SEQ ID NO: 146), NY-BR-1 (SEQ ID NO: 147), SURVIVIN (SEQ IDNO: 148), MAGE-A11 (SEQ ID NO: 149), PRAME (SEQ ID NO: 150), MAGE-A9(SEQ ID NO: 151), HOM-TES-85 (SEQ ID NO: 152), PIWIL-2 (SEQ ID NO: 349),EpCAM (SEQ ID NO: 154), HIWI (SEQ ID NO: 350), PLU-1 (SEQ ID NO: 351),TSGA10 (SEQ ID NO: 351), ODF-4 (SEQ ID NO: 352), SP17 (SEQ ID NO:354),RHOXF-2 (SEQ ID NO: 355), and NY-ESO-1 (SEQ ID NO: 356); one or moreovarian cancer-associated antigens selected from PIWIL-4 (SEQ ID NO:357), WT1 (SEQ ID NO: 358), EpCAM (SEQ ID NO: 154), BORIS (SEQ ID NO:145), AKAP-4 (SEQ ID NO: 144), OY-TES-1 (SEQ ID NO: 359), SP17 (SEQ IDNO: 354), PIWIL-2 (SEQ ID NO: 349), PIWIL-3 (SEQ ID NO: 360), SPAG9 (SEQID NO: 143), PRAME (SEQ ID NO: 150), HIWI (SEQ ID NO: 350), SURVIVIN(SEQ ID NO: 148), and AKAP-3 (SEQ ID NO: 361); and/or one or morecolorectal cancer-associated antigens selected from TSP50 (SEQ ID NO:153), EpCAM (SEQ ID NO: 154), SPAG9 (SEQ ID NO: 143), CAGE1 (SEQ ID NO:155), FBXO39 (SEQ ID NO: 156), SURVIVIN (SEQ ID NO: 148), MAGE-A8 (SEQID NO 157), MAGE-A6 (SEQ ID NO: 158), LEMD1 (SEQ ID NO:348) and MAGE-A3(SEQ ID NO: 347). In some cases the peptide comprises or consists of oneor more amino acid sequences selected from SEQ ID NOs: 41-80, or fromSEQ ID NOs: 41-80, 195-233, 251-271 and 302-331 that are optimised for Tcell activation/binding to all HLA types across the population.

In some cases the amino acid sequence is flanked at the N and/or Cterminus by additional amino acids that are not part of the sequence ofthe target polypeptide antigen, in other words that are not the samesequence of consecutive amino acids found adjacent to the selectedfragments in the target polypeptide antigen. In some cases the sequenceis flanked by up to 41 or 35 or 30 or 25 or 20 or 15 or 10, or 9 or 8 or7 or 6 or 5 or 4 or 3 or 2 or 1 additional amino acid at the N and/or Cterminus or between target polypeptide fragments. In other cases eachpolypeptide either consists of a fragment of a target polypeptideantigen, or consists of two or more such fragments arranged end to end(arranged sequentially in the peptide end to end) or overlapping in asingle peptide (where two or more of the fragments comprise partiallyoverlapping sequences, for example where two PEPIs in the samepolypeptide are within 50 amino acids of each other).

When fragments of different polypeptides or from different regions ofthe same polypeptide are joined together in an engineered peptide thereis the potential for neoepitopes to be generated around the join orjunction. Such neoepitopes encompass at least one amino acid from eachfragment on either side of the join or junction, and may be referred toherein as junctional amino acid sequences. The neoepitopes may induceundesired T cell responses against healthy cells (autoimmunity). Thepolypeptides may be designed, or the polypeptides may be screened, toavoid, eliminate or minimise neoepitopes that correspond to a fragmentof a protein expressed in normal healthy human cells and/or neoepitopesthat are capable of binding to at least two, or in some cases at leastthree, or at least four HLA class I molecules of the subject, or in somecases at least two, or at least three or four or five HLA class IImolecules of the subject. In some cases the peptide is designed, or thepolypeptide screened, to eliminate polypeptides having a junctionalneoepitope that is capable of binding in more than a thresholdpercentage of human subjects in an intent-to-treat population, to atleast two HLA class I molecules expressed by individual subjects of thepopulation. In some cases the threshold is 20%, or 15%, or 10%, or 5%,or 2%, or 1%, or 0.5% of said population. Alignment may be determinedusing known methods such as BLAST algorithms. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov/).

The presence in a vaccine or immunotherapy composition of at least twopolypeptide fragments (epitopes) that can bind to at least three HLAclass I of an individual (≥2 PEPI3+) is predictive for a clinicalresponse. In other words, if ≥2 PEPI3+ can be identified within theactive ingredient polypeptide(s) of a vaccine or immunotherapycomposition, then an individual is a likely clinical responder. The atleast two multiple HLA-binding PEPIs of the composition polypeptides mayboth target a single antigen (e.g a polypeptide vaccine comprising twomultiple HLA-binding PEPIs derived from a single tumor associatedantigen targeted by the vaccine) or may target different antigens (e.g.a polypeptide vaccine comprising one multiple HLA-binding PEPI derivedfrom one tumor associated antigen and a second multiple HLA-binding PEPIderived from a different tumor associated antigen).

Without wishing to be bound by theory, the inventors believe that onereason for the increased likelihood of deriving clinical benefit from avaccine/immunotherapy comprising at least two multiple-HLA bindingPEPIs, is that diseased cell populations, such as cancer or tumor cellsor cells infected by viruses or pathogens such as HIV, are oftenheterogenous both within and between effected subjects. A specificcancer patient, for example, may or may not express or overexpress aparticular cancer associated target polypeptide antigen of a vaccine, ortheir cancer may comprise heterogeneous cell populations, some of which(over-)express the antigen and some of which do not. In addition, thelikelihood of developing resistance is decreased when more multipleHLA-binding PEPIs are included or targeted by a vaccine/immunotherapybecause a patient is less likely to develop resistance to thecomposition through mutation of the target PEPI(s).

Currently most vaccines and immunotherapy compositions target only asingle polypeptide antigen. However according to the present disclosureit is in some cases beneficial to provide a pharmaceutical compositionthat targets two or more different polypeptide antigens. For example,most cancers or tumors are heterogeneous, meaning that different canceror tumor cells of a subject (over-)express different antigens. Thetumour cells of different cancer patients also express differentcombinations of tumour-associated antigens. The anti-cancer immunogeniccompositions that are most likely to be effective are those that targetmultiple antigens expressed by the tumor, and therefore more cancer ortumor cells, in an individual human subject or in a population.

The beneficial effect of combining multiple bestEPIs in a singletreatment (administration of one or more pharmaceutical compositionsthat together comprise multiple PEPIs), can be illustrated by thepersonalised vaccine polypeptides described in Examples 15 and 16 below.Exemplary CTA expression probabilities in ovarian cancer are as follows:BAGE: 30%; MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. If patient XYZwere treated with a vaccine comprising PEPIs in only BAGE and MAGE A9,then the probability of having a mAGP (multiple expressed antigens withPEPI) would be 11%. If patent XYZ were treated with a vaccine comprisingonly PEPIs for the MAGE A4 and MAGE A10 CTAs, then the probability ofhaving a multiAGP would be 19%. However if a vaccine contained all 4 ofthese CTAs (BAGE, MAGE A9, MAGE A4 and MAGE A10), then the probabilityof having a mAGP would be 50%. In other words the effect would begreater than the combined probabilities of mAGP for both two-PEPItreatments (probability mAGP for BAGE/MAGE+ probability mAGP for MAGE A4and MAGE A10). Patient XYZ's PIT vaccine described in Example 21contains a further 9 PEPIs, and thus, the probability of having a mAGPis over 99.95%.

Likewise exemplary CTA expression probabilities in breast cancer are asfollows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. Treatmentof patient ABC with a vaccine comprising PEPIs in only MAGE C2: 21% andMAGE A1 has a mAGP probability of 7%. Treatment of patient ABC with avaccine comprising PEPIs in only SPC1: 38%; MAGE A9 has a mAGPprobability of 11%. Treatment of patient ABC with a vaccine comprisingPEPIs in MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 has a mAGPprobability of 44% (44>7+11). Patient ABC's PIT vaccine described inExample 22 contains a further 8 PEPIs, and thus, the probability ofhaving a mAGP is over 99.93%.

Accordingly in some cases, the polypeptide or panel of polypeptides ofthe disclosure or an active ingredient polypeptide of a pharmaceuticalcomposition or kit of the disclosure may comprise or consist of anycombination of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 fragments of at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24 or 25 one or more of the cancer associated antigens, or CTAs,such as the CTA discussed above. Each fragment comprises or consists ofa different target epitope having an amino acid sequence selected fromSEQ ID NOs: 1-40; or selected from SEQ ID NOs: 1 to 20; or selected fromSEQ ID NOs: 21 to 40; or selected from SEQ ID NOs: 1-20, 24 and 172-194;or selected from SEQ ID NOs: 21-40 and 234-250; or selected from SEQ IDNOs: 272-301; or selected from SEQ ID NOs: 1-40, 172-194 and 234-250; orselected from SEQ ID NOs: 21-40, 234-250 and 272-301; or selected fromSEQ ID NOs: 1-20, 24, 172-194 and 272-301; or selected from SEQ ID NOs:1-40, 172-194, 234-250 and 272-301; or selected from SEQ ID NOs: 41-60,64 and 195-233; or selected from SEQ ID NOs: 61-80 and 251-271; orselected from SEQ ID NOs: 302-331; or selected from SEQ ID NOs: 41-80,195-233 and 251-271; or selected from SEQ ID NOs: 61-80, 251-271 and 302to 331; or selected from SEQ ID NOs: 41-60, 64, 191-233 and 302 to 331;or selected from SEQ ID NOs: 41-80, 195-233, 251-271 and 332-346; orselected from SEQ ID NOs: 1-20, 24, 41-60, 64, 172-194 and 195-233; orselected from SEQ ID NOs: 21-40, 61-80, 234-250 and 251-271; or selectedfrom SEQ ID NOs: 271-331; or selected from SEQ ID NOs: 1-80, 172-194,195-233, 234-250 and 251-271; or selected from SEQ ID NOs: 21-40, 61-80,234-250, 251-271, 272-301 and 302-331; or selected from SEQ ID NOs:1-80, 172-233, 234-271 and 272-331; or selected from SEQ ID NOs: 81-111and 435-449; or selected from SEQ ID NOs: 112-142; or selected from SEQID NOs: 332-346; or selected from SEQ ID NOs: 81-142; or selected fromSEQ ID NOs: 112-142 and 332-346; or selected from SEQ ID NOs: 81-111,435-449 and 332-346; or selected from SEQ ID NOs: 81-142 and 332-346; orselected from SEQ ID NOs: 41-60, 64, 81-111, 435-449 and 195-233; orselected from SEQ ID NOs: 61-80, 112-142 and 251-271; or selected fromSEQ ID NOs: 302-346; or selected from SEQ ID NOs: 41-142, 195-233 and251-271; or selected from SEQ ID NOs: 61-80, 112-142, 251-271 and302-346; or selected from SEQ ID NOs: 41-60, 64, 81-111, 435-449,195-233 and 302-346; or selected from SEQ ID NOs: 41-142, 195-233,251-271 and 302-346; or selected from SEQ ID NOs: 1-20, 24, 41-60, 64,81-111, 435-449 and 172-233; or selected from SEQ ID NOs: 21-40, 61-80,112-142, or 234-271; or selected from SEQ ID NOs: 272-346; or selectedfrom SEQ ID NOs: 1-142 and 172-271; or selected from SEQ ID NOs: 21-40,61-80, 112-142 and 234-346; or selected from SEQ ID NOs: 1-20, 24,41-60, 64, 81-111, 435-449, 172-233 and 272 to 346; or selected from SEQID NOs: 1-142 and 172-346; or selected from SEQ ID NOs: 1 to 2, or to 3,or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14,or 15, or 16, or 17, or 18, or 19, or SEQ ID NOs: 20 to 21, or to 22, or23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or33, or 34, or 35, or 36, or 37, or 38, or 39; or a different amino acidsequences selected from SEQ ID NOs: 41 to 80, or SEQ ID NOs: 41 to 60,or SEQ ID NOs: 61-80; or SEQ ID NOs: 41 to 42, or to 43, or to 44, or to45, or to 46, or to 47, or to 48, or to 49, or 50, or 51, or 52, or 53,or 54, or 55, or 56, or 57, or 58, or 59, SEQ ID NOs: 60 to 61, or to62, or to 63, or to 64, or to 65, or to 66, or to 67, or to 68, or to69, or to 70, or to 71, or to 72, or to 73, or to 74, or to 75, or to76, or to 77, or to 78, or to 79; a different amino acid sequencesselected from SEQ ID NOs: 81 to 142; or selected from SEQ ID NOs: 81 to82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 105, 106, 107, 108, 109, 110, or 111; or selectedfrom SEQ ID NOs: 81 to 105; or selected from SEQ ID NOs: 99, 100, 92,93, 101, 103, 104, 105 and 98; or selected from SEQ ID NOs: 112 to 142;or selected from SEQ ID NOs: 112 to 113, 114, 115, 116, 117, 118, 119,120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141 or 142; or selected from SEQ ID NOs:112 to 134; or selected from SEQ ID NOs: 121, 124, 126, 127, 130, 131,132, 133 and 134; or selected from SEQ ID NOs: 1 to 2, or to 3, or 4, or5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or16, or 17, or 18, or 19, or SEQ ID NOs: 20 to 21, or to 22, or 23, or24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or34, or 35, or 36, or 37, or 38, or 39; or a different amino acidsequences selected from SEQ ID NOs: 41 to 80, or SEQ ID NOs: 41 to 60,or SEQ ID NOs: 61-80; or SEQ ID NOs: 41 to 42, or to 43, or to 44, or to45, or to 46, or to 47, or to 48, or to 49, or 50, or 51, or 52, or 53,or 54, or 55, or 56, or 57, or 58, or 59, SEQ ID NOs: 60 to 61, or to62, or to 63, or to 64, or to 65, or to 66, or to 67, or to 68, or to69, or to 70, or to 71, or to 72, or to 73, or to 74, or to 75, or to76, or to 77, or to 78, or to 79; a different amino acid sequencesselected from SEQ ID NOs: 81 to 142; or selected from SEQ ID NOs: 81 to82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 105, 106, 107, 108, 109, 110, or 111; or selectedfrom SEQ ID NOs: 81 to 105; or selected from SEQ ID NOs: 99, 100, 92,93, 101, 103, 104, 105 and 98; or selected from SEQ ID NOs: 112 to 142;or selected from SEQ ID NOs: 112 to 113, 114, 115, 116, 117, 118, 119,120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141 or 142; or selected from SEQ ID NOs:112 to 134; or selected from SEQ ID NOs: 121, 124, 126, 127, 130, 131,132, 133 and 134; or selected from SEQ ID Nos: 130, 121, 131, 124, 134,126; or selected from SEQ ID NO: 435-449; or selected from any of thesegroups of sequences excluding SEQ ID NOs: 12, 32, 19 and/or 39, and/orSEQ ID NOs: 21, 41, 23 and/or 43 and/or SEQ ID NOs: 172, 177, 195 and/or203, and/or SEQ ID NOs: 1, 41 and/or 197, and/or SEQ ID NOs: 4, 44and/or 201, and/or SEQ ID NOs: 1, 4, 44, 197 and/or 201, and/or SEQ IDNOs: 1, 41, 197, 184 and/or 212, and/or SEQ ID NOs: 3, 43 and/or 200,and/or SEQ ID NOs: 3, 43, 200, 7 and/or 47, and/or SEQ ID NOs: 10, 50and/or 220, and/or SEQ ID NOs: 24, 64 and/or 202, and/or SEQ ID NOs: 6,46 and/or 209, and/or SEQ ID NOs: 182, 210, 185 and/or 213, and/or SEQID NOs: 14, 54, 225 and 226, and/or SEQ ID NOs: 190, 218, 11, 51 and/or219, and/or SEQ ID NOs: 12, 224 and/or 52, and/or SEQ ID NOs:192, 227and/or 228, and/or SEQ ID NOs:17, 229, 230 and/or 57, and/or SEQ ID NOs:21, 252, 61 and/or 253, and/or SEQ ID NOs: 23, 63 and/or 256, and/or SEQID NOs: 21, 252, 61, 253, 23, 63 and/or 256, and/or SEQ ID NOs: 237and/or 238, and/or SEQ ID NOs: 26 and/or 240, and/or SEQ ID NOs: 242,244, 263 and/or 265, and/or SEQ ID NOs: 29, 69 and/or 259, and/or SEQ IDNOs: 24, 64 and/or 255, and/or SEQ ID NOs: 236, 257 and/or 258, and/orSEQ ID NOs: 27, 67, 241 and/or 262, and/or SEQ ID NOs: 252, 249 and/or264, and/or SEQ ID NOs: 35, 250 and/or 75, and/or SEQ ID NOs: 252, 249,264, 35, 250 and/or 75, and/or SEQ ID NOs: 36, 266 and/or 76, and/or SEQID NOs: 36, 266, 76, 39 and/or 79, and/or SEQ ID NOs: 38, 268 and/or 78,and/or SEQ ID NOs: 38, 268, 78, 246 and/or 270, and/or SEQ ID NOs: 245,269, and/or 248, and/or SEQ ID NOs: 245, 269, 248, 40 and/or 80, and/orSEQ ID NOs: 272, 302, 281 and/or 311, and/or SEQ ID NOs: 276, 306, 300and/or 330, and/or SEQ ID NOs: 276, 306, 289 and/or 319, and/or SEQ IDNOs: 277, 307, 283 and/or 313, and/or SEQ ID NOs: 277, 307, 290 and/or320, and/or SEQ ID NOs: 282, 312, 297 and/or 327, or any othercombinations of the sequences disclosed herein that are within 50-60amino acids of each other in any one or more of the antigens of SEQ IDNOs: 143-158 and 347 to 351; and/or SEQ ID NOs: 18, 19 and/or 20 and/orSEQ ID NOs: 34-40; and/or SEQ ID NOs corresponding to peptides shown inTable 17, 20 and/or 23 having a N %*B % value of less than 12% or 13% or14% or 17.6% or 17.8% or 18% or 20% or 21% or 22% or 22.2% or 24% or 25%or 27% or 28% or 30% or 31% or 31.5% or 32% or 32.5% or 35%. In somecases the panel of peptides comprises or consists of one or morepolypeptides comprising or consisting of the amino acid sequences of SEQID NOs: 130, 121, 131, 124, 134, 126 and/or SEQ ID NOs: 435-449.

In some cases the disclosure provides a panel of any two or more of thepeptides or groups of peptides described above. For example the panelmay comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25 or more such peptides. In some cases thepanel comprises or consists of peptides comprising or consisting of allor any combination of the amino acid sequences of SEQ ID NOs: 99, 100,92, 93, 101, 103, 104, 105 and 98; or the amino acid sequences of SEQ IDNOs: 121, 124, 126, 127, 130, 131, 132, 133 and 134. In some cases thepanel comprises or consists of peptides comprising or consisting of allor any combination of the amino acid sequences of SEQ ID NOs: SEQ IDNOs: 130, 121, 131, 124, 134, 126 and/or SEQ ID NOs: 435-449.

Pharmaceutical Compositions, Methods of Treatment and Modes ofAdministration

In some aspects the disclosure relates to a pharmaceutical composition,kit, or panels of polypeptides as described above having one or morepolypeptides as active ingredient(s). These may be for use in a methodof inducing an immune response, treating, vaccinating or providingimmunotherapy to a subject, and the pharmaceutical composition may be avaccine or immunotherapy composition. Such a treatment comprisesadministering one or more polypeptides or pharmaceutical compositionsthat together comprise all of the active ingredient polypeptides of thetreatment to the subject. Multiple polypeptides or pharmaceuticalcompositions may be administered together or sequentially, for exampleall of the pharmaceutical compositions or polypeptides may beadministered to the subject within a period of 1 year, or 6 months, or 3months, or 60 or 50 or 40 or 30 days.

The term “active ingredient” as used herein refers to a polypeptide thatis intended to induce an immune response and may include a polypeptideproduct of a vaccine or immunotherapy composition that is produced invivo after administration to a subject. For a DNA or RNA immunotherapycomposition, the polypeptide may be produced in vivo by the cells of asubject to whom the composition is administered. For a cell-basedcomposition, the polypeptide may be processed and/or presented by cellsof the composition, for example autologous dendritic cells or antigenpresenting cells pulsed with the polypeptide or comprising an expressionconstruct encoding the polypeptide. The pharmaceutical composition maycomprise a polynucleotide or cell encoding one or more active ingredientpolypeptides.

The composition/kit may optionally further comprise at least onepharmaceutically acceptable diluent, carrier, or preservative and/oradditional polypeptides that do not comprise any PEPIs. The polypeptidesmay be engineered or non-naturally occurring. The kit may comprise oneor more separate containers each containing one or more of the activeingredient peptides. The composition/kit may be a personalised medicineto prevent, diagnose, alleviate, treat, or cure a disease of anindividual, such as a cancer.

The immunogenic or pharmaceutical compositions or kits described hereinmay comprise, in addition to one or more immunogenic peptides, apharmaceutically acceptable excipient, carrier, diluent, buffer,stabiliser, preservative, adjuvant or other materials well known tothose skilled in the art. Such materials are preferably non-toxic andpreferably do not interfere with the pharmaceutical activity of theactive ingredient(s). The pharmaceutical carrier or diluent may be, forexample, water containing solutions. The precise nature of the carrieror other material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intradermal, and intraperitoneal routes.

The pharmaceutical compositions of the disclosure may comprise one ormore “pharmaceutically acceptable carriers”. These are typically large,slowly metabolized macromolecules such as proteins, saccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose(WO 00/56365), lactose and lipid aggregates (such as oil droplets orliposomes). Such carriers are well known to those of ordinary skill inthe art. The pharmaceutical compositions may also contain diluents, suchas water, saline, glycerol, etc. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present. Sterile pyrogen-free, phosphate bufferedphysiologic saline is a typical carrier (Gennaro, 2000, Remington: TheScience and Practice of Pharmacy, 20th edition, ISBN:0683306472).

The pharmaceutical compositions of the disclosure may be lyophilized orin aqueous form, i.e. solutions or suspensions. Liquid formulations ofthis type allow the compositions to be administered direct from theirpackaged form, without the need for reconstitution in an aqueous medium,and are thus ideal for injection. The pharmaceutical compositions may bepresented in vials, or they may be presented in ready filled syringes.The syringes may be supplied with or without needles. A syringe willinclude a single dose, whereas a vial may include a single dose ormultiple doses.

Liquid formulations of the disclosure are also suitable forreconstituting other medicaments from a lyophilized form. Where apharmaceutical composition is to be used for such extemporaneousreconstitution, the disclosure provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with thecontents of the syringe being used to reconstitute the contents of thevial prior to injection.

The pharmaceutical compositions of the disclosure may include anantimicrobial, particularly when packaged in a multiple dose format.Antimicrobials may be used, such as 2-phenoxyethanol or parabens(methyl, ethyl, propyl parabens). Any preservative is preferably presentat low levels. Preservative may be added exogenously and/or may be acomponent of the bulk antigens which are mixed to form the composition(e.g. present as a preservative in pertussis antigens).

The pharmaceutical compositions of the disclosure may comprise detergente.g. Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF(dimethylformamide). Detergents are generally present at low levels,e.g. <0.01%, but may also be used at higher levels, e.g. 0.01-50%.

The pharmaceutical compositions of the disclosure may include sodiumsalts (e.g. sodium chloride) and free phosphate ions in solution (e.g.by the use of a phosphate buffer).

In certain embodiments, the pharmaceutical composition may beencapsulated in a suitable vehicle either to deliver the peptides intoantigen presenting cells or to increase the stability. As will beappreciated by a skilled artisan, a variety of vehicles are suitable fordelivering a pharmaceutical composition of the disclosure. Non-limitingexamples of suitable structured fluid delivery systems may includenanoparticles, liposomes, microemulsions, micelles, dendrimers and otherphospholipid-containing systems. Methods of incorporating pharmaceuticalcompositions into delivery vehicles are known in the art.

In order to increase the immunogenicity of the composition, thepharmacological compositions may comprise one or more adjuvants and/orcytokines.

Suitable adjuvants include an aluminum salt such as aluminum hydroxideor aluminum phosphate, but may also be a salt of calcium, iron or zinc,or may be an insoluble suspension of acylated tyrosine, or acylatedsugars, or may be cationically or anionically derivatised saccharides,polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A(MPL), lipid A derivatives (e.g. of reduced toxicity), 3-O-deacylatedMPL [3D-MPL], quil A, Saponin, QS21, Freund's Incomplete Adjuvant (DifcoLaboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.), AS-2 (Smith-Kline Beecham, Philadelphia, Pa.), CpGoligonucleotides, bioadhesives and mucoadhesives, microparticles,liposomes, polyoxyethylene ether formulations, polyoxyethylene esterformulations, muramyl peptides or imidazoquinolone compounds (e.g.imiquamod and its homologues). Human immunomodulators suitable for useas adjuvants in the disclosure include cytokines such as interleukins(e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colonystimulating factor (M-CSF), tumour necrosis factor (TNF), granulocyte,macrophage colony stimulating factor (GM-CSF) may also be used asadjuvants.

In some embodiments, the compositions comprise an adjuvant selected fromthe group consisting of Montanide ISA-51 (a water-in-oil emulsion,Seppic, Inc., Fairfield, N.J., United States of America), QS-21 (AquilaBiopharmaceuticals, Inc., Lexington, Mass., United States of America),GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacteriumparvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene(DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete andincomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin,pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheriatoxin (DT).

By way of example, the cytokine may be selected from the groupconsisting of a transforming growth factor (TGF) such as but not limitedto TGF-α and TGF-β; insulin-like growth factor-I and/or insulin-likegrowth factor-II; erythropoietin (EPO); an osteoinductive factor; aninterferon such as but not limited to interferon-α,-β, and -γ; a colonystimulating factor (CSF) such as but not limited to macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF). In some embodiments, the cytokine is selected from the groupconsisting of nerve growth factors such as NGF-β; platelet-growthfactor; a transforming growth factor (TGF) such as but not limited toTGF-α. and TGF-β; insulin-like growth factor-I and insulin-like growthfactor-II; erythropoietin (EPO); an osteoinductive factor; an interferon(IFN) such as but not limited to IFN-α, IFN-β, and IFN-γ; a colonystimulating factor (CSF) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); aninterleukin (Il) such as but not limited to IL-1, IL-1.alpha., IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18; LIF; kit-ligand or FLT-3;angiostatin; thrombospondin; endostatin; a tumor necrosis factor (TNF);and LT.

It is expected that an adjuvant or cytokine can be added in an amount ofabout 0.01 mg to about 10 mg per dose, preferably in an amount of about0.2 mg to about 5 mg per dose. Alternatively, the adjuvant or cytokinemay be at a concentration of about 0.01 to 50%, preferably at aconcentration of about 2% to 30%.

In certain aspects, the pharmaceutical compositions of the disclosureare prepared by physically mixing the adjuvant and/or cytokine with thepeptides of the disclosure under appropriate sterile conditions inaccordance with known techniques to produce the final product.

Examples of suitable compositions of the invented polypeptide fragmentsand methods of administration are provided in Esseku and Adeyeye (2011)and Van den Mooter G. (2006). Vaccine and immunotherapy compositionpreparation is generally described in Vaccine Design (“The subunit andadjuvant approach” (eds Powell M. F. & Newman M. J. (1995) Plenum PressNew York). Encapsulation within liposomes, which is also envisaged, isdescribed by Fullerton, U.S. Pat. No. 4,235,877.

In some embodiments, the compositions disclosed herein are prepared as anucleic acid vaccine. In some embodiments, the nucleic acid vaccine is aDNA vaccine. In some embodiments, DNA vaccines, or gene vaccines,comprise a plasmid with a promoter and appropriate transcription andtranslation control elements and a nucleic acid sequence encoding one ormore polypeptides of the disclosure. In some embodiments, the plasmidsalso include sequences to enhance, for example, expression levels,intracellular targeting, or proteasomal processing. In some embodiments,DNA vaccines comprise a viral vector containing a nucleic acid sequenceencoding one or more polypeptides of the disclosure. In additionalaspects, the compositions disclosed herein comprise one or more nucleicacids encoding peptides determined to have immunoreactivity with abiological sample. For example, in some embodiments, the compositionscomprise one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptidescomprising a fragment that is a T cell epitope capable of binding to atleast three HLA class I molecules and/or at least three HLA class IImolecules of a patient. In some embodiments, the peptides are derivedfrom an antigen that is expressed in cancer. In some embodiments the DNAor gene vaccine also encodes immunomodulatory molecules to manipulatethe resulting immune responses, such as enhancing the potency of thevaccine, stimulating the immune system or reducing immunosuppression.Strategies for enhancing the immunogenicity of of DNA or gene vaccinesinclude encoding of xenogeneic versions of antigens, fusion of antigensto molecules that activate T cells or trigger associative recognition,priming with DNA vectors followed by boosting with viral vector, andutilization of immunomodulatory molecules. In some embodiments, the DNAvaccine is introduced by a needle, a gene gun, an aerosol injector, withpatches, via microneedles, by abrasion, among other forms. In some formsthe DNA vaccine is incorporated into liposomes or other forms ofnanobodies. In some embodiments, the DNA vaccine includes a deliverysystem selected from the group consisting of a transfection agent;protamine; a protamine liposome; a polysaccharide particle; a cationicnanoemulsion; a cationic polymer; a cationic polymer liposome; acationic nanoparticle; a cationic lipid and cholesterol nanoparticle; acationic lipid, cholesterol, and PEG nanoparticle; a dendrimernanoparticle. In some embodiments, the DNA vaccines is administered byinhalation or ingestion. In some embodiments, the DNA vaccine isintroduced into the blood, the thymus, the pancreas, the skin, themuscle, a tumor, or other sites.

In some embodiments, the compositions disclosed herein are prepared asan RNA vaccine. In some embodiments, the RNA is non-replicating mRNA orvirally derived, self-amplifying RNA. In some embodiments, thenon-replicating mRNA encodes the peptides disclosed herein and contains5′ and 3′ untranslated regions (UTRs). In some embodiments, the virallyderived, self-amplifying RNA encodes not only the peptides disclosedherein but also the viral replication machinery that enablesintracellular RNA amplification and abundant protein expression. In someembodiments, the RNA is directly introduced into the individual. In someembodiments, the RNA is chemically synthesized or transcribed in vitro.In some embodiments, the mRNA is produced from a linear DNA templateusing a T7, a T3, or an Sp6 phage RNA polymerase, and the resultingproduct contains an open reading frame that encodes the peptidesdisclosed herein, flanking UTRs, a 5′ cap, and a poly(A) tail. In someembodiments, various versions of 5′ caps are added during or after thetranscription reaction using a vaccinia virus capping enzyme or byincorporating synthetic cap or anti-reverse cap analogues. In someembodiments, an optimal length of the poly(A) tail is added to mRNAeither directly from the encoding DNA template or by using poly(A)polymerase. The RNA encodes one or more peptides comprising a fragmentthat is a T cell epitope capable of binding to at least three HLA classI and/or at least three HLA class II molecules of a patient. In someembodiments, the fragments are derived from an antigen that is expressedin cancer. In some embodiments, the RNA includes signals to enhancestability and translation. In some embodiments, the RNA also includesunnatural nucleotides to increase the half-life or modified nucleosidesto change the immunostimulatory profile. In some embodiments, the RNAsis introduced by a needle, a gene gun, an aerosol injector, withpatches, via microneedles, by abrasion, among other forms. In some formsthe RNA vaccine is incorporated into liposomes or other forms ofnanobodies that facilitate cellular uptake of RNA and protect it fromdegradation. In some embodiments, the RNA vaccine includes a deliverysystem selected from the group consisting of a transfection agent;protamine; a protamine liposome; a polysaccharide particle; a cationicnanoemulsion; a cationic polymer; a cationic polymer liposome; acationic nanoparticle; a cationic lipid and cholesterol nanoparticle; acationic lipid, cholesterol, and PEG nanoparticle; a dendrimernanoparticle; and/or naked mRNA; naked mRNA with in vivoelectroporation; protamine-complexed mRNA; mRNA associated with apositively charged oil-in-water cationic nanoemulsion; mRNA associatedwith a chemically modified dendrimer and complexed with polyethyleneglycol (PEG)-lipid; protamine-complexed mRNA in a PEG-lipidnanoparticle; mRNA associated with a cationic polymer such aspolyethylenimine (PEI); mRNA associated with a cationic polymer such asPEI and a lipid component; mRNA associated with a polysaccharide (forexample, chitosan) particle or gel; mRNA in a cationic lipidnanoparticle (for example, 1,2-dioleoyloxy-3-trimethylammoniumpropane(DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNAcomplexed with cationic lipids and cholesterol; or mRNA complexed withcationic lipids; cholesterol and PEG-lipid. In some embodiments, the RNAvaccine is administered by inhalation or ingestion. In some embodiments,the RNA is introduced into the blood, the thymus, the pancreas, theskin, the muscle, a tumor, or other sites, and/or by an intradermal,intramuscular, subcutaneous, intranasal, intranodal, intravenous,intrasplenic, intratumoral or other delivery route.

Polynucleotide or oligonucleotide components may be naked nucleotidesequences or be in combination with cationic lipids, polymers ortargeting systems. They may be delivered by any available technique. Forexample, the polynucleotide or oligonucleotide may be introduced byneedle injection, preferably intradermally, subcutaneously orintramuscularly. Alternatively, the polynucleotide or oligonucleotidemay be delivered directly across the skin using a delivery device suchas particle-mediated gene delivery. The polynucleotide oroligonucleotide may be administered topically to the skin, or to mucosalsurfaces for example by intranasal, oral, or intrarectal administration.

Uptake of polynucleotide or oligonucleotide constructs may be enhancedby several known transfection techniques, for example those includingthe use of transfection agents. Examples of these agents includecationic agents, for example, calcium phosphate and DEAE-Dextran andlipofectants, for example, lipofectam and transfectam. The dosage of thepolynucleotide or oligonucleotide to be administered can be altered.

Administration is typically in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to resultin a clinical response or to show clinical benefit to the individual,e.g. an effective amount to prevent or delay onset of the disease orcondition, to ameliorate one or more symptoms, to induce or prolongremission, or to delay relapse or recurrence.

The dose may be determined according to various parameters, especiallyaccording to the substance used; the age, weight and condition of theindividual to be treated; the route of administration; and the requiredregimen. The amount of antigen in each dose is selected as an amountwhich induces an immune response. A physician will be able to determinethe required route of administration and dosage for any particularindividual. The dose may be provided as a single dose or may be providedas multiple doses, for example taken at regular intervals, for example2, 3 or 4 doses administered hourly. Typically peptides, polynucleotidesor oligonucleotides are typically administered in the range of 1 pg to 1mg, more typically 1 pg to 10 μg for particle mediated delivery and 1 μgto 1 mg, more typically 1-100 more typically 5-50 μg for other routes.Generally, it is expected that each dose will comprise 0.01-3 mg ofantigen. An optimal amount for a particular vaccine can be ascertainedby studies involving observation of immune responses in subjects.

Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams & Wilkins.

In some cases in accordance with the disclosure, more than one peptideor composition of peptides is administered. Two or more pharmaceuticalcompositions may be administered together/simultaneously and/or atdifferent times or sequentially. Thus, the disclosure includes sets ofpharmaceutical compositions and uses thereof. The use of combination ofdifferent peptides, optionally targeting different antigens, isimportant to overcome the challenges of genetic heterogeneity of tumorsand HLA heterogeneity of individuals. The use of peptides of thedisclosure in combination expands the group of individuals who canexperience clinical benefit from vaccination. Multiple pharmaceuticalcompositions of peptides of the disclosure, manufactured for use in oneregimen, may define a drug product.

Routes of administration include but are not limited to intranasal,oral, subcutaneous, intradermal, and intramuscular. The subcutaneousadministration is particularly preferred. Subcutaneous administrationmay for example be by injection into the abdomen, lateral and anterioraspects of upper arm or thigh, scapular area of back, or upperventrodorsal gluteal area.

The compositions of the disclosure may also be administered in one, ormore doses, as well as, by other routes of administration. For example,such other routes include, intracutaneously, intravenously,intravascularly, intraarterially, intraperitnoeally, intrathecally,intratracheally, intracardially, intralobally, intramedullarly,intrapulmonarily, and intravaginally. Depending on the desired durationof the treatment, the compositions according to the disclosure may beadministered once or several times, also intermittently, for instance ona monthly basis for several months or years and in different dosages.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

One or more compositions of the disclosure may be administered, or themethods and uses for treatment according to the disclosure may beperformed, alone or in combination with other pharmacologicalcompositions or treatments, for example chemotherapy and/orimmunotherapy and/or vaccine. The other therapeutic compositions ortreatments may for example be one or more of those discussed herein, andmay be administered either simultaneously or sequentially with (beforeor after) the composition or treatment of the disclosure.

In some cases the treatment may be administered in combination withcheckpoint blockade therapy, co-stimulatory antibodies, chemotherapyand/or radiotherapy, targeted therapy or monoclonal antibody therapy. Ithas been demonstrated that chemotherapy sensitizes tumors to be killedby tumor specific cytotoxic T cells induced by vaccination (Ramakrishnanet al. J Clin Invest. 2010; 120(4):1111-1124). Examples for checkpointinhibitors are CTLA-4 inhibitor, Ipilimumab and programmed celldeath-1/programmed cell death ligand-1 (PD-1/PD-L1) signalinginhibitors, Nibolumab, Pembrolizumab, Atezolizumab and Durvalumab.Examples of chemotherapy agents include alkylating agents includingnitrogen mustards such as mechlorethamine (HN2), cyclophosphamide,ifosfamide, melphalan (L-sarcolysin) and chlorambucil; anthracyclines;epothilones; nitrosoureas such as carmustine (BCNU), lomustine (CCNU),semustine (methyl-CCNU) and streptozocin (streptozotocin); triazenessuch as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide;ethylenimines/methylmelamines such as hexamethylmelamine, thiotepa;alkyl sulfonates such as busulfan; Antimetabolites including folic acidanalogues such as methotrexate (amethopterin); alkylating agents,antimetabolites, pyrimidine analogs such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) andcytarabine (cytosine arabinoside); purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG) and pentostatin (2′-deoxycoformycin);epipodophylotoxins; enzymes such as L-asparaginase; biological responsemodifiers such as IFNα, IL-2, G-CSF and GM-CSF; platinum coordinationcomplexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin;anthracenediones such as mitoxantrone and anthracycline; substitutedurea such as hydroxyurea; methylhydrazine derivatives includingprocarbazine (N-methylhydrazine, MIH) and procarbazine; adrenocorticalsuppressants such as mitotane (o,p′-DDD) and aminoglutethimide; taxoland analogues/derivatives; hormones and agonists/antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide, progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate, estrogen such as diethylstilbestrol and ethinyl estradiolequivalents, antiestrogen such as tamoxifen, androgens includingtestosterone propionate and fluoxymesterone/equivalents, antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs and leuprolideand non-steroidal antiandrogens such as flutamide; natural productsincluding vinca alkaloids such as vinblastine (VLB) and vincristine,epipodophyllotoxins such as etoposide and teniposide, antibiotics suchas dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin),doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin(mitomycin C), enzymes such as L-asparaginase, and biological responsemodifiers such as interferon alphenomes.

In some cases the method of treatment is a method of vaccination or amethod of providing immunotherapy. As used herein, “immunotherapy” isthe prevention or treatment of a disease or condition by inducing orenhancing an immune response in an individual. In certain embodiments,immunotherapy refers to a therapy that comprises the administration ofone or more drugs to an individual to elicit T cell responses. In aspecific embodiment, immunotherapy refers to a therapy that comprisesthe administration or expression of polypeptides that contain one ormore PEPIs to an individual to elicit a T cell response to recognize andkill cells that display the one or more PEPIs on their cell surface inconjunction with a class I HLAs. In another specific embodiment,immunotherapy comprises the administration of one or more PEPIs to anindividual to elicit a cytotoxic T cell response against cells thatdisplay tumor associated antigens (TAAs) or cancer testis antigens(CTAs) comprising the one or more PEPIs on their cell surface. Inanother embodiment, immunotherapy refers to a therapy that comprises theadministration or expression of polypeptides that contain one or morePEPIs presented by class II HLAs to an individual to elicit a T helperresponse to provide co-stimulation to cytotoxic T cells that recognizeand kill diseased cells that display the one or more PEPIs on their cellsurface in conjunction with a class I HLAs. In still another specificembodiment, immunotherapy refers to a therapy that comprisesadministration of one or more drugs to an individual that reactivateexisting T cells to kill target cells. The theory is that the cytotoxicT cell response will eliminate the cells displaying the one or morePEPIs, thereby improving the clinical condition of the individual. Insome instances, immunotherapy may be used to treat tumors. In otherinstances, immunotherapy may be used to treat intracellularpathogen-based diseases or disorders.

In some cases the disclosure relates to the treatment of cancer or thetreatment of solid tumors. In some cases the treatment is of breastcancer, ovarian cancer or colorectal cancer. In other cases thetreatment may be of any other cancer or solid tumor that expresses atarget tumor associated antigen of the present peptides as describedherein, or any cancer in which such target polypeptide antigens areexpressed in some or a high percentage of subjects. The treatment may beof cancers or malignant or benign tumors of any cell, tissue, or organtype. The cancer may or may not be metastatic. Exemplary cancers includecarcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, orblastomas. The cancer may or may not be a hormone related or dependentcancer (e.g., an estrogen or androgen related cancer).

Selection of Polypeptides and Patients

Specific polypeptide antigens, and particularly short peptides derivedfrom such antigens that are commonly used in vaccination andimmunotherapy, induce immune responses in only a fraction of humansubjects. The polypeptides of the present disclosure are specificallyselected to induce immune responses in a high proportion of the generalpopulation, but they may not be effective in all individuals due to HLAgenotype heterogeneity. HLA genotype population heterogeneity means thatthe immune or clinical response rate to the vaccines described hereinwill differ between different human subpopulations. In some cases thevaccines described herein are for use to treat a specific or targetsubpopulation, for example an Asian population, or a Vietnamese,Chinese, and/or Japanese population.

The disclosure also provides a method of identifying a human subject whowill likely have a cytotoxic T cell response to administration of apharmaceutical composition comprising a peptide of the disclosure(likely responders), or of predicting the likelihood that a subject willhave a cytotoxic T cell response.

As provided herein T cell epitope presentation by multiple HLAs of anindividual is generally needed to trigger a T cell response. The bestpredictor of a cytotoxic T cell response to a given polypeptide, asdetermined by the inventors, is the presence of at least one T cellepitope that is presented by three or more HLA class I of an individual(≥1 PEPI3+). Accordingly the presence within the active ingredientpeptides of a pharmaceutical composition of one or more T cell epitopesthat is capable of binding to at least three HLA of a subject ispredictive for the subject having a cytotoxic T cell response toadministration of the pharmaceutical composition. The subject is alikely immune responder.

In some cases the T cell epitope that is capable of binding to at leastthree HLA class I of the subject has the amino acid sequence of any oneof SEQ ID NOs: 1 to 40, or SEQ ID NOs: 1 to 40, 172-194, 234-250 and272-301. In other cases the T cell epitope may have a different aminoacid sequence within the one or more peptides of the pharmaceuticalcomposition.

The inventors have further discovered that the presence in a vaccine orimmunotherapy composition of at least two epitopes that can bind to atleast three HLA of an individual is predictive for a clinical response.In other words, if an individual has a total of ≥2 PEPI3+ within theactive ingredient polypeptide(s) of a vaccine or immunotherapycomposition, and these PEPI3+s are derived from antigen sequences thatare in fact expressed in the individual (for example, target tumor cellsof the individual express the target tumor-associated antigens), thenthe individual is a likely clinical responder (i.e. a clinicallyrelevant immune responder).

Accordingly some aspects of the disclosure relate to a method ofidentifying a subject who will likely have a clinical response to amethod of treatment according to the disclosure, or of predicting thelikelihood that a subject will have a clinical response. A “clinicalresponse” or “clinical benefit” as used herein may be the prevention ora delay in the onset of a disease or condition, the amelioration of oneor more symptoms, the induction or prolonging of remission, or the delayof a relapse or recurrence or deterioration, or any other improvement orstabilisation in the disease status of a subject. Where appropriate, a“clinical response” may correlate to “disease control” or an “objectiveresponse” as defined by the Response Evaluation Criteria In Solid Tumors(RECIST) guidelines.

In some embodiments the method comprises determining that one or morecancer-associated antigens selected from SPAG9, AKAP-4, BORIS,NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-85,TSP50, EpCAM, CAGE1, FBXO39, MAGE-A8 and MAGE-A6 is expressed by acancer. For example expression of the cancer associated antigen may bedetected in a sample obtained from the subject, for example a tumorbiopsy, using methods that are known in the art.

The inventors have discovered that it is not sufficient that a vaccineor immunotherapy composition targets an antigen that is expressed bycancer or tumor cells of a patient, nor that the target sequences ofthat antigen can bind to HLA class I of the patient (HLA restrictedepitopes). The composition is likely effective only in patients thatboth express the target antigen and have three or more HLA class I thatbind to a single T cell epitope of the target antigen. Moreover, asdescribed above, at least two epitopes that binds to at least 3 HLAs ofthe patient are generally needed to induce a clinically relevant immuneresponse.

Therefore the method further comprises determining that the activeingredient peptide(s) of the pharmaceutical composition comprise two ormore different amino acid sequences each of which is a) a fragment of acancer-associated antigen expressed by cancer cells of the subject,determined as described above; and b) a T cell epitope capable ofbinding to at least three HLA class I of the subject.

In some cases the T cell epitope that is capable of binding to at leastthree HLA class I of the subject has the amino acid sequence of any oneof SEQ ID NOs: 1 to 40, or SEQ ID NOs: 1 to 40, 172-194, 234-250 and272-301. In other cases the T cell epitope may have a different aminoacid sequence within the one or more peptides of the pharmaceuticalcomposition.

In some cases the likelihood that a subject will have a clinicalresponse to a peptide vaccine or immunotherapy composition, such asthose described herein, can be determined without knowing whether thetarget antigens are expressed in cancer or tumor cells of the subjectand/or without determining the HLA class I genotype of the subject.Known antigen expression frequencies in the disease (e.g. MAGE-A3 in atumor type like breast or colorectal cancer) and/or known frequenciesfor HLA class I and class II genotype of subjects in the targetpopulation (e.g ethnic population, general population, diseasedpopulation) may be used instead. Moreover by combining peptides thattarget the most frequently presented PEPIs across the population(BestEPIs) in multiple frequently expressed target antigens in thedisease, as identified and described herein, it is possible to design acancer vaccine regime that is effective for a high proportion ofpatients. However, using the companion diagnostic methods describedherein to pre-select patients who are most likely to have a clinicalresponse will increase clinical response rates amongst treated patients.

The likelihood that a subject will respond to treatment is increased by(i) the presence of more multiple HLA-binding PEPIs in the activeingredient polypeptides; (ii) the presence of PEPIs in more targetpolypeptide antigens; and (iii) expression of the target polypeptideantigens in the subject or in diseased cells of the subject. In somecases expression of the target polypeptide antigens in the subject maybe known, for example if target polypeptide antigens are in a sampleobtained from the subject. In other cases, the probability that aspecific subject, or diseased cells of a specific subject,(over-)express a specific or any combination of target polypeptideantigens may be determined using population expression frequency data,e.g. probability of expression of an antigen in breast cancer,colorectal cancer or ovarian cancer. The population expression frequencydata may relate to a subject- and/or disease-matched population or theintent-to-treat population. For example, the frequency or probability ofexpression of a particular cancer-associated antigen in a particularcancer or subject having a particular cancer, for example breast cancer,can be determined by detecting the antigen in tumor, e.g. breast cancertumor samples. In some cases such expression frequencies may bedetermined from published figures and scientific publications. In somecases a method of the disclosure comprises a step of determining theexpression frequency of a relevant target polypeptide antigen in arelevant population.

Disclosed is a range of pharmacodynamic biomarkers to predict theactivity/effect of vaccines in individual human subjects as well as inpopulations of human subjects. These biomarkers expedite more effectivevaccine development and also decrease the development cost and may beused to assess and compare different compositions. Exemplary biomarkersare as follows.

-   -   AG95—potency of a vaccine: The number of antigens in a cancer        vaccine that a specific tumor type expresses with 95%        probability. AG95 is an indicator of the vaccine's potency, and        is independent of the immunogenicity of the vaccine antigens.        AG95 is calculated from the tumor antigen expression rate data.        Such data may be obtained from experiments published in peer        reviewed scientific journals. Technically, AG95 is determined        from the binomial distribution of antigens in the vaccine, and        takes into account all possible variations and expression rates.    -   PEPI3+ count—immunogenicity of a vaccine in a subject:        Vaccine-derived PEPI3+ are personal epitopes that bind to et        least 3 HLAs of a subject and induce T cell responses. PEPI3+        can be determined using the PEPI3+ Test in subjects who's        complete 4-digit HLA genotype is known.    -   AP count—antigenicity of a vaccine in a subject: Number of        vaccine antigens with PEPI3+. Vaccines contain sequences from        target polypeptide antigens expressed by diseased cells. AP        count is the number of antigens in the vaccine that contain        PEPI3+, and the AP count represents the number of antigens in        the vaccine that can induce T cell responses in a subject. AP        count characterizes the vaccine-antigen specific T cell        responses of the subject since it depends only on the HLA        genotype of the subject and is independent of the subject's        disease, age, and medication. The correct value is between 0 (no        PEPI presented by the antigen) and maximum number of antigens        (all antigens present PEPIs).    -   AP50—antigenicity of a vaccine in a population: The mean number        of vaccine antigens with a PEPI in a population. The AP50 is        suitable for the characterization of vaccine-antigen specific T        cell responses in a given population since it depends on the HLA        genotype of subjects in a population.    -   AGP count—effectiveness of a vaccine in a subject: Number of        vaccine antigens expressed in the tumor with PEPI. The AGP count        indicates the number of tumor antigens that vaccine recognizes        and induces a T cell response against (hit the target). The AGP        count depends on the vaccine-antigen expression rate in the        subject's tumor and the HLA genotype of the subject. The correct        value is between 0 (no PEPI presented by expressed antigen) and        maximum number of antigens (all antigens are expressed and        present a PEPI).    -   AGP50—effectiveness of a cancer vaccine in a population: The        mean number of vaccine antigens expressed in the indicated tumor        with PEPI (i.e., AGP) in a population. The AGP50 indicates the        mean number of tumor antigens that the T cell responses induced        by the vaccine can recognize. AGP50 is dependent on the        expression rate of the antigens in the indicated tumor type and        the immunogenicity of the antigens in the target population.        AGP50 can estimate a vaccine's effectiveness in different        populations and can be used to compare different vaccines in the        same population. The computation of AGP50 is similar to that        used for AG50, except the expression is weighted by the        occurrence of the PEPI3+ in the subject on the expressed vaccine        antigens. In a theoretical population, where each subject has a        PEPI from each vaccine antigen, the AGP50 will be equal to AG50.        In another theoretical population, where no subject has a PEPI        from any vaccine antigen, the AGP50 will be 0. In general, the        following statement is valid: 0≤AGP50≤AG50.    -   mAGP—a candidate biomarker for the selection of likely        responders: Likelihood that a cancer vaccine induces T cell        responses against multiple antigens expressed in the indicated        tumor. mAGP is calculated from the expression rates of        vaccine-antigens in the tumor and the presence of vaccine        derived PEPIs in the subject. Technically, based on the AGP        distribution, the mAGP is the sum of probabilities of the        multiple AGP (≥2 AGPs).

The results of a prediction as set out above may be used to inform aphysician's decisions concerning treatment of the subject. Accordingly,in some cases the method of the disclosure predicts that a subject willhave or is likely to have a T cell response and/or a clinical responseto a treatment as described herein, and the method further comprisesselecting the treatment for the human subject. In some cases a subjectis selected for treatment if their likelihood of a response targeted ata predefined number of target polypeptide antigens, optionally whereinthe target polypeptide antigens are (predicted to be) expressed, isabove a predetermined threshold. In some cases the number of targetpolypeptide antigens or epitopes is two. In some cases the number oftarget polypeptide antigens or epitopes is three, or four, or five, orsix, or seven, or eight, or nine, or ten. The method may furthercomprise administering the treatment to the human subject.Alternatively, the method may predict that the subject will not have animmune response and/or a clinical response and further compriseselecting a different treatment for the subject.

Further Embodiments of the Disclosure

1. A polypeptide that comprises a fragment of up to 50 consecutive aminoacids of

(a) a colorectal cancer-associated antigen selected from TSP50, EpCAM,SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6 and MAGE-A3,wherein the fragment comprises an amino acid sequence selected from anyone of SEQ ID NOs: 21 to 40 and 234 to 250;

(b) an ovarian cancer-associated antigen selected from PIWIL-4, WT1,EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME,HIWI, SURVIVIN, and AKAP-3 wherein the fragment comprises the amino acidsequence of any one of SEQ ID NOs: 272 to 301; and/or

(c) a breast cancer associated antigen selected from PIWIL-2, AKAP-4,EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME,NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1wherein the fragment comprises an amino acid sequence selected from anyone of SEQ ID NOs: 1 to 20, 24 and 172 to 194;

optionally wherein the fragment is flanked at the N and/or C terminus byadditional amino acids that are not part of the sequence of the breast,ovarian or colorectal cancer-associated antigen.

2. The polypeptide of item 1, wherein the polypeptide

-   -   a. is a fragment of a colorectal cancer-associated antigen        selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN,        MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein the fragment        comprises an amino acid sequence selected from any one of SEQ ID        NOs: 21 to 40 and 234 to 250; or    -   b. comprises or consists of two or more fragments of one or more        colorectal cancer associated antigens selected from TSP50,        EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3        and LEMD1, wherein each fragment comprises a different amino        acid sequence selected from any one of SEQ ID NOs: 21 to 40 and        234 to 250, optionally wherein the fragments overlap or are        arranged end to end in the polypeptide; or    -   c. is a fragment of a ovarian cancer-associated antigen selected        from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17,        PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3,        wherein the fragment comprises an amino acid sequence selected        from any one of SEQ ID NOs: 272 to 301; or    -   d. comprises or consists of two or more fragments of one or more        ovarian cancer associated antigens selected from PIWIL-4, WT1,        EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9,        PRAME, HIWI, SURVIVIN and AKAP-3, wherein each fragment        comprises a different amino acid sequence selected from any one        of SEQ ID NOs: 272 to 301, optionally wherein the fragments        overlap or are arranged end to end in the polypeptide; or    -   e. is a fragment of a breast cancer associated antigen selected        from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN,        MAGE-A11, PRAME, MAGE-A9, HOM-TES-85, PIWIL-2, EpCAM, HIWI,        PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein the fragment        comprises the amino acid sequence from any one of SEQ ID NOs: 1        to 20, 24 and 172 to 194; or    -   f. comprises or consists of two or more fragments of one or more        breast cancer associated antigens selected from SPAG9, AKAP-4,        BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9,        HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17,        RHOXF-2, wherein each fragment comprises a different amino acid        sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and        172 to 194; optionally wherein the fragments overlap or are        arranged end to end in the polypeptide and.

3. The polypeptide according to item 1 or item 2, wherein thepolypeptide comprises or consists of fragments of at least two differentcancer-associated antigens, wherein the cancer-associated antigens areselected from

-   -   (a) TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8,        MAGE-A6, MAGE-A3 and LEMD1;    -   (b) PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2,        PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3; and/or    -   (c) SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN,        MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI,        PLU-1, TSGA10, ODF-4, SP17, RHOXF-2;        wherein each fragment comprises a different amino acid sequence        selected from SEQ ID NOs: 21 to 40 and 234 to 250; SEQ ID NOs:        272 to 301; and/or SEQ ID NOs: 1 to 20, 24 and 172 to 194.

4. The polypeptide according to any one of items 1 to 3, comprising orconsisting of one or more amino acid sequences selected from SEQ ID NOs:41-80, 251 to 271, 302 to 331 and 196 to 233.

5. The polypeptide according to any one of items 1 to 4 comprising orconsisting of the amino acid sequence of any one of SEQ ID NOs: 81 to142, 332 to 346 and 435-449.

6. A panel of two or more polypeptides according to any one of items 1to 5, wherein

-   -   (a) each polypeptide comprises a different amino acid sequence        selected from SEQ ID NOs: 21 to 40 and 234 to 250; or    -   (b) each polypeptide comprises a different amino acid sequence        selected from SEQ ID NOs: 272 to 301; or    -   (c) each peptide comprises a different amino acid sequence        selected from SEQ ID NOs: 1 to 20, 24 and 172 to 194; or (c)        each peptide comprises a different amino acid sequence selected        from SEQ ID NOs: 1 to 40, 234 to 250, 272 to 301 and 172 to 194.

7. The panel of polypeptides according to item 6 comprising six peptideshaving the amino acid sequences of SEQ ID NOs: 130, 121, 131, 124, 134,126.

8. A pharmaceutical composition or kit having one or more polypeptidesaccording to any one of items 1 to 5, or a panel of polypeptidesaccording to item 6 or item 7, or a polypeptide comprising at least twoamino acid sequences selected SEQ ID NOs: 21 to 40 and 234 to 250; SEQID NOs: 272 to 301; and/or SEQ ID NOs: 1 to 20, 24 and 172 to 194 as anactive ingredient.

9. A method of vaccination, providing immunotherapy or inducing acytotoxic T cell response in a subject, the method comprisingadministering to the subject a pharmaceutical composition according toitem 8.

10. The method of item 9 that is a method of treating cancer, optionallycolorectal cancer, ovarian cancer or breast cancer.

11. A method of identifying a human subject who will likely have acytotoxic T cell response to administration of a pharmaceuticalcomposition according to item 8, the method comprising

-   -   (i) determining that the active ingredient polypeptide(s) of the        pharmaceutical composition comprise a sequence that is a T cell        epitope capable of binding to at least three HLA class I        molecules of the subject; and    -   (ii) identifying the subject as likely to have a cytotoxic T        cell response to administration of the pharmaceutical        composition.

12. The method of item 11 further comprising using population expressiondata for each antigen that

-   -   (a) is selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39,        SURVIVIN, LEMD1, MAGE-A8, MAGE-A6, MAGE-A3, PIWIL-4, WT1, BORIS,        AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, PRAME, HIWI, PLU-1,        TSGA10, ODF-4, RHOXF-2, NY-SAR-35, MAGE-A9, NY-BR-1, MAGE-A11,        HOM-TES-85, NY-ESO-1 and AKAP-3; and    -   (b) comprises an amino acid sequence that is        -   i. a fragment of an active ingredient peptide of the            pharmaceutical composition; and        -   ii. a T cell epitope capable of binding to at least three            HLA class I molecules of the subject;    -   to determine the likelihood that the subject will have a        cytotoxic T cell response that targets one or more polypeptide        antigens that are expressed by cancer cells of the subject.

13. A method of identifying a subject who will likely have a clinicalresponse to a method of treatment according to item 10, the methodcomprising

-   -   (i) determining that the active ingredient polypeptide(s) of the        pharmaceutical composition comprise two or more different amino        acid sequences each of which is        -   a. a T cell epitope capable of binding to at least three HLA            class I molecules of the subject; and        -   b. a fragment of a cancer-associated antigen expressed by            cancer cells of the subject, optionally wherein the            cancer-associated antigen is present in a sample obtained            from the subject; and    -   (ii) identifying the subject as likely to have a clinical        response to the method of treatment.

14. A method of determining the likelihood that a specific human subjectwill have a clinical response to a method of treatment according to item10, wherein one or more of the following factors corresponds to a higherlikelihood of a clinical response:

-   -   (a) presence in the active ingredient polypeptide(s) of a higher        number of amino acid sequences and/or different amino acid        sequences that are each a T cell epitope capable of binding to        at least three HLA class I of the subject;    -   (b) a higher number of target polypeptide antigens, comprising        at least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; optionally wherein the target            polypeptide antigens are expressed in the subject, further            optionally wherein the target polypeptides antigens are in            one or more samples obtained from the subject;    -   (c) a higher probability that the subject expresses target        polypeptide antigens, optionally a threshold number of the        target polypeptide antigens and/or optionally target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; and/or    -   (d) a higher number of target polypeptide antigens that the        subject is predicted to express, optionally a higher number of        target polypeptide antigens that the subject expresses with a        threshold probability, and/or optionally the target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject.

15. The method of item 14, wherein the method comprises

-   -   (i) identifying which polypeptide antigens targeted by the        active ingredient polypeptide(s) comprise an amino acid sequence        that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject;    -   (ii) using population expression data for each antigen        identified in step (i) to determine the probability that the        subject expresses one or more of the antigens identified in        step (i) that together comprise at least two different amino        acid sequences of step (i); and    -   (iii) determining the likelihood that the subject will have a        clinical response to administration of the pharmaceutical        composition, kit or panel of polypeptides, wherein a higher        probability determined in step (ii) corresponds to a more likely        clinical response.

16. The method of item 15, wherein the at least two different amino acidsequences are comprised in the amino acid sequence of two differentpolypeptide antigens targeted by the active ingredient polypeptide(s).

The method of any one of items 13 to 16 further comprising selecting orrecommending administration of the pharmaceutical composition as amethod of treatment for the subject, and optionally further treating thesubject by administering the pharmaceutical composition.

A method of treatment according to item 10, wherein the subject has beenidentified as likely to have a clinical response or as having above athreshold minimum likelihood of having a clinical response to thetreatment by a method according to any one of items 13 to 16.

The method of any one of items 9, 10, 17 and 18 wherein the treatment isadministered in combination with chemotherapy, targeted therapy or acheckpoint inhibitor.

A method of identifying a human subject who will likely not have aclinical response to a method of treatment according to item 10, themethod comprising

-   -   (i) determining that the active ingredient peptide(s) of the        pharmaceutical composition do not comprise two or more different        amino acid sequences each of which is a T cell epitope capable        of binding to at least three HLA class I molecules of the        subject; and    -   (iii) identifying the subject as likely not to have a clinical        response to the method of treatment.

EXAMPLES Example 1—HLA-Epitope Binding Prediction Process and Validation

Predicted binding between particular HLA and epitopes (9 mer peptides)was based on the Immune Epitope Database tool for epitope prediction(www.iedb.org).

The HLA I-epitope binding prediction process was validated by comparisonwith HLA I-epitope pairs determined by laboratory experiments. A datasetwas compiled of HLA I-epitope pairs reported in peer reviewedpublications or public immunological databases.

The rate of agreement with the experimentally determined dataset wasdetermined (Table 2). The binding HLA I-epitope pairs of the datasetwere correctly predicted with a 93% probability. Coincidentally thenon-binding HLA I-epitope pairs were also correctly predicted with a 93%probability.

TABLE 2 Analytical specificity and sensitivity of the HLA-epitopebinding prediction process. True epitopes False epitopes (n = 327) (n =100) HLA-epitope pairs (Binder match) (Non-binder match) HIV  91% (32)82% (14) Viral 100% (35) 100% (11)  Tumor  90% (172) 94% (32) Other(fungi, bacteria, etc.) 100% (65) 95% (36) All  93% (304) 93% (93)

The accuracy of the prediction of multiple HLA binding epitopes wasdetermined. Based on the analytical specificity and sensitivity usingthe 93% probability for both true positive and true negative predictionand 7% (=100%-93%) probability for false positive and false negativeprediction, the probability of the existence of a multiple HLA bindingepitope in a person can be calculated. The probability of multiple HLAbinding to an epitope shows the relationship between the number of HLAsbinding an epitope and the expected minimum number of real binding. PerPEPI definition three is the expected minimum number of HLA to bind anepitope (bold).

TABLE 3 Accuracy of multiple HLA binding epitopes predictions. Expectedminimum number of real Predicted number of HLAs binding to an epitopeHLA binding 0 1 2 3 4 5 6 1 35%  95%  100%  100%  100%  100% 100% 2 6%29%  90%  99% 100%  100% 100% 3 1% 4% 22%  84% 98% 100% 100% 4 0% 0% 2%16% 78%  96%  99% 5 0% 0% 0%  1% 10%  71%  94% 6 0% 0% 0%  0%  0%  5% 65%

The validated HLA-epitope binding prediction process was used todetermine all HLA-epitope binding pairs described in the Examples below.

Example 2—Epitope Presentation by Multiple HLA Predicts Cytotoxic TLymphocyte (CTL) Response

The presentation of one or more epitopes of a polypeptide antigen by oneor more HLA I of an individual is predictive for a CTL response wasdetermined.

The study was carried out by retrospective analysis of six clinicaltrials, conducted on 71 cancer and 9 HIV-infected patients (Table 4)¹⁻⁷.Patients from these studies were treated with an HPV vaccine, threedifferent NY-ESO-1 specific cancer vaccines, one HIV-1 vaccine and aCTLA-4 specific monoclonal antibody (Ipilimumab) that was shown toreactivate CTLs against NY-ESO-1 antigen in melanoma patients. All ofthese clinical trials measured antigen specific CD8+ CTL responses(immunogenicity) in the study subjects after vaccination. In some cases,correlation between CTL responses and clinical responses were reported.

No patient was excluded from the retroactive study for any reason otherthan data availability. The 157 patient datasets (Table 4) wererandomized with a standard random number generator to create twoindependent cohorts for training and evaluation studies. In some casesthe cohorts contained multiple datasets from the same patient, resultingin a training cohort of 76 datasets from 48 patients and atest/validation cohort of 81 datasets from 51 patients.

TABLE 4 Summary of patient datasets Immunoassay # Data sets performed inHLA Clinical Target # (#antigen × the clinical genotyping trialImmunotherapy Antigen Disease Patients* #patient) trials** method Ref 1VGX-3100 HPV16-E6 Cervical 17/18 5 × 17 IFN-γ High 1 HPV16-E7 cancerELISPOT Resolution HPV18-E6 SBT HPV18-E7 HPV16/18 2 HIVIS vaccine HIV-1Gag AIDS  9/12 2 × 9  IFN-γ Low-Medium 2 HIV-1 RT ELISPOT Resolution SSO3 rNY-ESO-1 NY-ESO-1 Breast-and 18/18 1 × 18 In vitro and High 3 ovarianEx vivo IFN-γ Resolution 4 cancers, ELISPOT SBT melanoma and sarcoma 4Ipilimumab NY-ESO-1 Metastatic 19/20 1 × 19 ICS after Low to medium 5melanoma T-cell resolution stimulation typing, SSP of genomic DNA, highresolution sequencing 5 NY-ESO-1f NY-ESO-1 Esophageal-, 10/10 1 × 10 ICSafter SSO probing 6 (91-110) non-small- T-cell and SSP of cell lung-stimulation genomic DNA and gastric cancer 6 NY-ESO-1 NY-ESO-1Esophageal- 7/9 1 × 7  ICS after SSO probing 7 overlapping (79-173) andlung T-cell and SSP of peptides cancer, stimulation genomic DNAmalignant melanoma Total 6 7 80 157 N/A *Number of patients used in theretrospective analysis from the original number of patient of theclinical trials. **Immunoassays are based on T cell stimulation withantigen-specific peptide pools and quantify the released cytokines bydifferent techniques. CT: Clinical trial; SBT: Sequence Based Typing;SSO: Sequence-Specific Oligonucleotide; ICS: Intracellular cytokinestaining; SSP: Sequence-specific priming

The reported CTL responses of the training dataset were compared withthe HLA I restriction profile of epitopes (9 mers) of the vaccineantigens. The antigen sequences and the HLA I genotype of each patientwere obtained from publicly available protein sequence databases or peerreviewed publications and the HLA I-epitope binding prediction processwas blinded to patients' clinical CTL response data. The number ofepitopes from each antigen predicted to bind to at least 1 (PEPI1+), orat least 2 (PEPI2+), or at least 3 (PEPI3+), or at least 4 (PEPI4+), orat least 5 (PEPI5+), or all 6 (PEPI6) HLA class I molecules of eachpatient was determined and the number of HLA bound were used asclassifiers for the reported CTL responses. The true positive rate(sensitivity) and true negative rate (specificity) were determined fromthe training dataset for each classifier (number of HLA bound)separately.

ROC analysis was performed for each classifier. In a ROC curve, the truepositive rate (Sensitivity) was plotted in function of the falsepositive rate (1-Specificity) for different cut-off points (FIG. 1 ).Each point on the ROC curve represents a sensitivity/specificity paircorresponding to a particular decision threshold (epitope (PEPI) count).The area under the ROC curve (AUC) is a measure of how well theclassifier can distinguish between two diagnostic groups (CTL responderor non-responder).

The analysis unexpectedly revealed that predicted epitope presentationby multiple class I HLAs of a subject (PEPI2+, PEPI3+, PEPI4+, PEPI5+,or PEPI6), was in every case a better predictor of CTL response thanepitope presentation by merely one or more HLA class I (PEPI1+,AUC=0.48, Table 5).

TABLE 5 Determination of diagnostic value of the PEPI biomarker by ROCanalysis Classifiers AUC PEPI1+ 0.48 PEPI2+ 0.51 PEPI3+ 0.65 PEPI4+ 0.52PEPI5+ 0.5 PEPI6+ 0.5

The CTL response of an individual was best predicted by considering theepitopes of an antigen that could be presented by at least 3 HLA class Iof an individual (PEPI3+, AUC=0.65, Table 5). The threshold count ofPEPI3+(number of antigen-specific epitopes presented by 3 or more HLA ofan individual) that best predicted a positive CTL response was 1 (Table6). In other words, at least one antigen-derived epitope is presented byat least 3 HLA class I of a subject (≥1 PEPI3+), then the antigen cantrigger at least one CTL clone, and the subject is a likely CTLresponder. Using the ≥1 PEPI3+ threshold to predict likely CTLresponders (“≥1 PEPI3+ Test”) provided 76% diagnostic sensitivity (Table12).

TABLE 6 Determination of the ≥1 PEPI3+ threshold to predict likely CTLresponders in the training dataset. PEPI3+ Count 1 2 3 4 5 6 7 8 9 10 1112 Sensitivity: 0.76 0.60 0.31 0.26 0.14 0.02 0 0 0 0 0 0 1- 0.59 0.240.21 0.15 0.09 0.06 0.06 0.03 0.03 0.03 0.03 0.03

Example 3—Validation of the ≥1 PEPI3+ Test

The test cohort of 81 datasets from 51 patients was used to validate the≥1 PEPI3+ threshold to predict an antigen-specific CTL response. Foreach dataset in the test cohort it was determined whether the ≥1 PEPI3+threshold was met (at least one antigen-derived epitope presented by atleast three class I HLA of the individual). This was compared with theexperimentally determined CTL responses reported from the clinicaltrials (Table 7).

The clinical validation demonstrated that a PEPI3+ peptide induce CTLresponse in an individual with 84% probability. 84% is the same valuethat was determined in the analytical validation of the PEPI3+prediction, epitopes that binds to at least 3 HLAs of an individual(Table 3). These data provide strong evidences that immune responses areinduced by PEPIs in individuals.

TABLE 7 Diagnostic performance characteristics of the ≥1 PEPI3+ Test (n= 81). Performance characteristic Description Result Positive100%[A/(A + B)] The likelihood that an individual that meets the ≥1 84%predictive PEPI3+ threshold has antigen-specific CTL value (PPV)responses after treatment with immunotherapy. Sensitivity 100%[A/(A +C)] The proportion of subjects with antigen-specific 75% CTL responsesafter treatment with immunotherapy who meet the ≥1 PEPI3+ threshold.Specificity 100%[D/(B + D)] The proportion of subjects withoutantigen-specific 55% CTL responses after treatment with immunotherapywho do not meet the ≥1 PEPI3+ threshold. Negative 100%[D/(C + D)] Thelikelihood that an individual who does not meet 42% predictive the ≥1PEPI3+ threshold does not have antigen- value (NPV) specific CTLresponses after treatment with immunotherapy. Overall 100%[(A + D)/N]The percentage of predictions based on the ≥1 70% percent PEPI3+threshold that match the experimentally agreement (OPA) determinedresult, whether positive or negative. Fisher's exact (p) 0.01

ROC analysis determined the diagnostic accuracy, using the PEPI3+ countas cut-off values (FIG. 2 ). The AUC value=0.73. For ROC analysis an AUCof 0.7 to 0.8 is generally considered as fair diagnostic.

A PEPI3+ count of at least 1 (≥1 PEPI3+) best predicted a CTL responsein the test dataset (Table 8). This result confirmed the thresholddetermined during the training (Table 5).

TABLE 8 Confirmation of the ≥1 PEPI3+ threshold to predict likely CTLresponders in the test/validation dataset. PEPI3+ Count 1 2 3 4 5 6 7 89 10 11 12 Sensitivity: 0.75 0.52 0.26 0.23 0.15 0.13 0.08 0.05 0 0 0 01-Specificity: 0.45 0.15 0.05 0 0 0 0 0 0 0 0 0

Example 4—the ≥1 PEPI3+ Test Predicts CD8+ CTL Reactivities

The ≥1 PEPI3+ Test was compared with a previously reported method forpredicting a specific human subject's CTL response to peptide antigens.

The HLA genotypes of 28 cervical cancer and VIN-3 patients that receivedthe HPV-16 synthetic long peptide vaccine (LPV) in two differentclinical trials were determined from DNA samples⁸ ⁸ ⁹ ¹⁰. The LPVconsists of long peptides covering the HPV-16 viral oncoproteins E6 andE7. The amino acid sequence of the LPV was obtained from thesepublications. The publications also report the T cell responses of eachvaccinated patient to pools of overlapping peptides of the vaccine.

For each patient epitopes (9 mers) of the LPV that are presented by atleast three patient class I HLA (PEPI3+s) were identified and theirdistribution among the peptide pools was determined. Peptides thatcomprised at least one PEPI3+(≥1 PEPI3+) were predicted to induce a CTLresponse. Peptides that comprised no PEPI3+ were predicted not to inducea CTL response.

The ≥1 PEPI3+ Test correctly predicted 489 out of 512 negative CTLresponses and 8 out of 40 positive CTL responses measured aftervaccination (FIG. 3A). Overall, the agreement between the ≥1 PEPI3+ Testand experimentally determined CD8+ T cell reactivity was 90% (p<0.001).

For each patient the distribution among the peptide pools of epitopesthat are presented by at least one patient class I HLA (≥1 PEPI1+, HLArestricted epitope prediction, prior art method) was also determined. ≥1PEPI1+ correctly predicted 116 out of 512 negative CTL responses and 37out of 40 positive CTL responses measured after vaccination (FIG. 3B).Overall, the agreement between the HLA restricted epitope prediction (≥1PEPI1+) and CD8+ T cell reactivity was 28% (not significant).

Example 5—Prediction of HLA Class II Restricted CD4+ Helper T CellEpitopes

The 28 cervical cancer and VIN-3 patients that received the HPV-16synthetic long peptide vaccine (LPV) in two different clinical trials(as detailed in Example 4) were investigated for CD4+T helper responsesfollowing LPV vaccination (FIGS. 4A-B). The sensitivity of theprediction of HLA class II restricted epitopes was 78%, since the Stateof Art tool predicted 84 positive responses (positive CD4+ T cellreactivity to a peptide pool for a person's DP alleles) out of 107(sensitivity=78%). The specificity was 22% since it could rule out 7negative responses out of 31. Overall, the agreement betweenHLA-restricted class II epitope prediction and CD4+ T cell reactivitywas 66%, which was statistically not significant.

Example 6—the ≥1 PEPI3+ Test Predicts T Cell Responses to Full LengthLPV Polypeptides

Using the same reported studies as Examples 4 and 5, the ≥1 PEPI3+ Testwas used to predict patient CD8+ and CD4+ T cell responses to the fulllength E6 and E7 polypeptide antigens of the LPV vaccine. Results werecompared to the experimentally determined responses were reported. TheTest correctly predicted the CD8+ T cell reactivity (PEPI3+) of 11 outof 15 VIN-3 patients with positive CD8+ T cell reactivity test results(sensitivity 73%, PPV 85%) and of 2 out of 5 cervical cancer patients(sensitivity 40%, PPV 100%). The CD4+ T cell reactivities (PEPI4+) werecorrectly predicted 100% both of VIN-3 and cervical cancer patients(FIG. 5 ).

Class I and class II HLA restricted PEPI3+ count was also observed tocorrelate with the reported clinical benefit to LPV vaccinated patients.Patients with higher PEPI3+ counts had either complete or partialresponse already after 3 months.

Example 7—Case Study

pGX3001 is an HPV16 based DNA vaccine containing full length E6 and E7antigens with a linker in between. pGX3002 is an HPV18 based DNA vaccinecontaining full length E6 and E7 antigens with a linker in between. APhase II clinical trial investigated the T cell responses of 17HPV-infected patients with cervical cancer who were vaccinated with bothpGX3001 and pGX3002 (VGX-3100 vaccination)′.

FIGS. 5A-D and FIG. 6 shows for two illustrative patients (patient 12-11and patient 14-5) the position of each epitope (9 mer) presented by atleast 1 (PEPI1+), at least 2 (PEPI2+), at least 3 (PEPI3+), at least 4(PEPI4+), at least 5 (PEPI5+), or all 6 (PEPI6) class I HLA of thesepatients within the full length sequence of the two HPV-16 and twoHPV-18 antigens.

Patient 12-11 had an overall PEPI1+ count of 54 for the combinedvaccines (54 epitopes presented by one or more class I HLA). Patient14-5 had a PEPI1+ count of 91. Therefore patient 14-5 has a higherPEPI1+ count than patient 12-11 with respect to the four HPV antigens.The PEPI1+s represent the distinct vaccine antigen specific HLArestricted epitope sets of patients 12-11 and 14-5. Only 27 PEPI1+s werecommon between these two patients.

For the PEPI3+ counts (number of epitopes presented by three or morepatient class I HLA), the results for patients 12-11 and 14-5 werereversed. Patient 12-11 had a PEPI3+ count of 8, including at least onePEPI3+ in each of the four HPV16/18 antigens. Patient 14-5 had a PEPI3+count of 0.

The reported immune responses of these two patients matched the PEPI3+counts, not the PEPI1+ counts. Patient 12-11 developed immune responsesto each of the four antigens post-vaccination as measured by ELISpot,whilst patient 14-5 did not develop immune responses to any of the fourantigens of the vaccines. A similar pattern was observed when the PEPI1+and PEPI3+ sets of all 17 patients in the trial were compared. There wasno correlation between the PEPI1+ count and the experimentallydetermined T cell responses reported from the clinical trial. However,correlation between the T cell immunity predicted by the ≥1 PEPI3+ Testand the reported T cell immunity was observed. The ≥1 PEPI3+ Testpredicted the immune responders to HPV DNA vaccine.

Moreover, the diversity of the patient's PEPI3+ set resembled thediversity of T cell responses generally found in cancer vaccine trials.Patients 12-3 and 12-6, similar to patient 14-5, did not have PEPI3+spredicting that the HPV vaccine could not trigger T cell immunity. Allother patients had at least one PEPI3 predicting the likelihood that theHPV vaccine can trigger T cell immunity. 11 patients had multiple PEPI3+predicting that the HPV vaccine likely triggers polyclonal T cellresponses. Patients 15-2 and 15-3 could mount high magnitude T cellimmunity to E6 of both HPV, but poor immunity to E7. Other patients 15-1and 12-11 had the same magnitude response to E7 of HPV18 and HPV16,respectively.

Example 8—Design of a Model Population for Conducting in Silico Trialsand Identifying Candidate Precision Vaccine Targets for Large Population

An in silico human trial cohort of 433 subjects with complete 4-digitHLA class I genotype (2×HLA-A*xx:xx; 2×HLA-B*xx:xx; 2×HLA-C*xx:xx) anddemographic information was compiled. This Model Population has subjectswith mixed ethnicity having a total of 152 different HLA alleles thatare representative for >85% of presently known allele G-groups.

A database of a “Big Population” containing 7,189 subjects characterizedwith 4-digit HLA genotype and demographic information was alsoestablished. The Big Population has 328 different HLA class I alleles.The HLA allele distribution of the Model Population significantlycorrelated with the Big Population (Table 9) (Pearson p<0.001).Therefore the 433 patient Model Population is representative for a 16times larger population.

The Model Population is representative for 85% of the human race asgiven by HLA diversity as well as HLA frequency.

TABLE 9 Statistical analysis of HLA distributions in “Model Population”vs. “Big Population”. Pearson R Group name 1 Group name 2 valueCorrelation P Value 433 Model 7,189 Big 0.89 Strong P < 0.001 PopulationPopulation

Example 9—in Silico Trials Based on the Identification of Multiple HLABinding Epitopes Predict the Reported T Cell Response Rates of ClinicalTrials

The objective of this study was to determine whether a model population,such as the one described in Example 8, may be used to predict CTLreactivity rates of vaccines, i.e. used in an in silico efficacy trials.

Twelve peptide vaccines derived from cancer antigens that induced T cellresponses in a subpopulation of subjects were identified from peerreviewed publications. These peptides have been investigated in clinicaltrials enrolling a total of 172 patients (4 ethnicities). T cellresponses induced by the vaccine peptides have been determined fromblood specimens and reported. The immune response rate as the percentageof study subjects with positive T cell responses measured in theclinical trials was determined (FIG. 7 ).

TABLE 10 Clinical trials conducted with peptide vaccines. Source PeptidePop. Peptide vaccines antigen length T cell assay (n) Ethnicity RefMMNLMQPKTQQTYTYD JUP 16mer Multimer 18 Canadian 12 stainingGRGSTTTNYLLDRDDYRNTSD ADA17 21mer Multimer 18 Canadian 12 stainingLKKGAADGGKLDGNAKLNRSLK BAP31 22mer Multimer 18 Canadian 12 stainingFPPKDDHTLKFLYDDNQRPYPP TOP2A 22mer Multimer 18 Canadian 12 stainingRYRKPDYTLDDGHGLLRFKST Abl-2 21mer Multimer 18 Canadian 12 stainingQRPPFSQLHRFLADALNT DDR1 18mer Multimer 18 Canadian 12 stainingALDQCKTSCALMQQHYDQTSCFSSP ITGB8 25mer Multimer 18 Canadian 12 stainingSTAPPAHGVTSAPDTRPAPGSTAPP MUC-1 25mer Proliferation 80 Canadian 13YLEPGPVTA gp100 9mer Tetramer 18 US 14 MTPGTQSPFFLLLLLTVLTVV MUC-1 21merCytotoxicity 10 Israeli 15 SSKALQRPV Bcr-Abl 9mer ELISPOT 4 US 16RMFPNAPYL WT-1 9mer Multimer 24 US 17 staining RMFPNAPYL (HLA-A*0201)WT-1 9mer Cytokine 18 CEU 18 staining

The 12 peptides were investigated with the ≥1 PEPI3+ Test in each of the433 subjects of the Model Population described in Example 8. The “≥1PEPI3+ Score” for each peptide was calculated as the proportion ofsubjects in the Model Population having at least one vaccine derivedepitope that could bind to at least three subject-specific HLA class I(≥1 PEPI3+). If the corresponding clinical trial stratified patients forHLA allele selected population, the Model Population was also filteredfor subjects with the respective allele(s) (Example: WT1, HLA-A*0201).

The experimentally determined response rates reported from the trialswere compared with the ≥1 PEPI3+ Scores. The Overall Percentage ofAgreements (OPA) were calculated on the paired data (Table 11). A linearcorrelation between ≥1 PEPI3+ Score and response rate (R²=0.77) wasobserved (FIG. 7 ). This result shows that the identification ofpeptides predicted to bind to multiple HLAs of an individual is usefulto predict in silico the outcome of clinical trials.

TABLE 11Comparison of ≥1 PEPI3+ Scores and CTL response rates of 12 peptidevaccines. ≥1 PEPI3+ Score* Source Response rate (Model Peptide vaccineantigen (Clinical Trials) Population) OPA MMNLMQPKTQQTYTYD JUP  0% 22%NA GRGSTTTNYLLDRDDYRNTSD ADA17 11% 18% 61% LKKGAADGGKLDGNAKLNRSLK BAP3111%  7% 64% FPPKDDHTLKFLYDDNQRPYPP TOP2A 11% 39% 28%RYRKPDYTLDDGHGLLRFKST Abl-2 17% 12% 71% QRPPFSQLHRFLADALNT DDR1 17%  5%29% ALDQCKTSCALMQQHYDQTSCFSSP ITGB8 28% 31% 90%STAPPAHGVTSAPDTRPAPGSTAPP MUC-1 20%  2% 10% YLEPGPVTA gp100 28%  4% 14%MTPGTQSPFFLLLLLTVLTVV MUC-1 90% 95% 95% SSKALQRPV Bcr-Abl  0%  0% 100% RMFPNAPYL WT-1 100%  78% 78% RMFPNAPYL (HLA-A*0201) WT-1 81% 61% 75% *%subjects in the Model Population with ≥1 vaccine derived PEPI3+

Example 10. In Silico Trials Based on the Identification of Multiple HLABinding Epitopes Predict the Reported T Cell Response Rates of ClinicalTrials II

Nineteen clinical trials with published immune response rates (IRR)conducted with peptide or DNA based vaccines were identified (Table 19).These trials involved 604 patients (9 ethnicities) and covered 38vaccines derived from tumor and viral antigens. Vaccine antigen specificCTL responses were measured in each study patient and the response ratein the clinical study populations was calculated and reported.

Each vaccine peptide of the 19 clinical trials was investigated with the≥1 PEPI3+ Test in each subject of the Model Population. The ≥1 PEPI3+Score for each peptide was calculated as the proportion of subjects inthe Model Population having at least one vaccine derived PEPI3+. Theexperimentally determined response rates reported from the trials werecompared with the PEPI Scores, as in Example 9 (Table 20). A linearcorrelation between the response rate and ≥1 PEPI3+ Score (R²=0.70) wasobserved (FIG. 8 ). This result confirms that the identification ofpeptides predicted to bind to multiple HLAs of an individual can predictT cell responses of subjects, and in silico trials can predict theoutcome of clinical trials.

TABLE 12 Response rates published in clinical trials. CTL Pop. Race/Immunotherapy Type assay (n) Ethnicity Ref. StimuVax peptide Prolif- 80Canadian 13 eration gp100 vaccine DNA Tetramer 18 US 14 IMA901 phase Ipeptide ELISPOT 64 CEU IMA901 phase II peptide Multimer 27 CEU 19staining ICT107 peptide ICC 15 US 20 ProstVac DNA ELISPOT 32 CEU87%, ²1Afr. Am. 12%, Hisp. 1% Synchrotope DNA Tetramer 26 US 22 TA2M MELITAC12.1 peptide ELISPOT 167 US 23 WT1 vaccine peptide Tetramer 22 Japanese24 Ipilimumab checkpoint ICC 19 US 5 (NY-ESO-1) inhibitor ** VGX-3100DNA ELISPOT 17 US 1 HIVIS-1 DNA ELISPOT 12 CEU98%, 2 Asian1%, Hisp. 1%ImMucin peptide Cytotoxicity 10 Israeli 15 NY-ESO-1 OLP peptideIFN-gamma 7 Japanese 7 GVX301 peptide Proliferation 14 CEU 25 WT1vaccine peptide ELISPOT 12 US 26 WT1 vaccine peptide ICC 18 CEU 18DPX-0907* peptide Multimer 18 Canadian 12 staining Melanoma peptideELISPOT 26 White 27 peptide vaccine

TABLE 13 Linear correlation between PEPI Score and response rate (R² =0.7). Clinical Trial ≥1 PEPI3+ Immunotherapy Response Rate Score* OPAStimuVax (failed to show 20%  2% 10% efficacy in Phase III) gp100vaccine 28%  4% 14% IMA901 phase I 74% 48% 65% IMA901 phase II 64% 48%75% ICT107 33% 52% 63% ProstVac 45% 56% 80% Synchrotope TA2M 46% 24% 52%MELITAC 12.1 49% 47% 96% WT1 vaccine 59% 78% 76% Ipilimumab (NY-ESO-1*)72% 84% 86% VGX-3100 78% 87% 90% HIVIS-1 80% 93% 86% ImMucin 90% 95% 95%NY-ESO-1 OLP 100%  84% 84% GVX301 64% 65% 98% WT1 vaccine 83% 80% 96%WT1 vaccine 81% 61% 75% DPX-0907 61% 58% 95% Melanoma peptide vaccine52% 42% 81% *% subjects in the Model Population with ≥1 vaccine derivedPEPI3+

Example 11—in Silico Trial Based on the Identification of Multiple HLABinding Epitopes in a Multi-Peptide Vaccine Predict the ReportedClinical Trial Immune Response Rate

IMA901 is a therapeutic vaccine for renal cell cancer (RCC) comprising 9peptides derived from tumor-associated peptides (TUMAPs) that arenaturally presented in human cancer tissue. A total of 96 HLA-A*02+subjects with advanced RCC were treated with IMA901 in two independentclinical studies (phase I and phase II). Each of the 9 peptides ofIMA901 have been identified in the prior art as HLA-A2-restrictedepitopes. Based on currently accepted standards, they are all strongcandidate peptides to boost T cell responses against renal cancer in thetrial subjects, because their presence has been detected in renal cancerpatients, and because the trial patients were specifically selected tohave at least one HLA molecule capable of presenting each of thepeptides.

For each subject in the Model population how many of the nine peptidesof the IMA901 vaccine were capable of binding to three or more HLA wasdetermined. Since each peptide in the IMA901 vaccine is a 9 mer thiscorresponds to the PEPI3+ count. The results were compared with theimmune response rates reported for the Phase I and Phase II clinicaltrials (Table 14).

TABLE 14 Immune Response Rates in the Model Population and in twoclinical trials to IMA901 Model Population Immune responses to (HLA-A2+)Phase I Phase II TUMAPs (n = 180) (n = 27)* (n = 64)* No peptide 39% 25%36%  1 peptide 34% 44% 38% ≥2 peptides 27% 29% 26% (MultiPEPI Score) ≥3peptides  3% ND  3% *No of patients evaluated for immune responses

The phase I and phase II study results show the variability of theimmune responses to the same vaccine in different trial cohorts.Overall, however, there was a good agreement between response ratespredicted by the ≥2 PEPI3+ Test and the reported clinical responserates.

In a retrospective analysis, the clinical investigators of the trialsdiscussed above found that subjects who responded to multiple peptidesof the IMA901 vaccine were significantly (p=0.019) more likely toexperience disease control (stable disease, partial response) thansubjects who responded only to one peptide or had no response. 6 of 8subjects (75%) who responded to multiple peptides experienced clinicalbenefit in the trial, in contrast to 14% and 33% of 0 and 1 peptideresponders, respectively. The randomized phase II trial confirmed thatimmune responses to multiple TUMAPs were associated with a longeroverall survival.

Since the presence of PEPIs accurately predicted responders to TUMAPs,clinical responders to IMA901 are likely patients who can present ≥2PEPIs from TUMAPs. This subpopulation is only 27% of HLA-A*02 selectedpatients, and according to the clinical trial result, 75% of thissubpopulation is expected to experience clinical benefit. The sameclinical results suggest that 100% of patients would experience clinicalbenefit if patient selection is based on ≥3 PEPIs from TUMAPs, albeitthis population would represent only 3% of the HLA-A*02 selected patientpopulation. These results suggest that the disease control rate (stabledisease or partial response) is between 3% and 27% in the patientpopulation which was investigated in the IMA901 clinical trials. In theabsence of complete response, only a portion of these patients canexperience survival benefit.

These findings explain the absence of improved survival in the Phase IIIIMA901 clinical trial. These results also demonstrated that HLA-A*02enrichment of the study population was not sufficient to reach theprimary overall survival endpoint in the Phase III IMA901 trial. As theIMA901 trial investigators noted, there is a need for the development ofa companion diagnostic (CDx) to select likely responders to peptidevaccines. These findings also suggest that selection of patients with ≥2TUMAP specific PEPIs may provide sufficient enrichment to demonstratesignificant clinical benefit of IMA901.

Example 12—in Silico Trial Based on the Identification ofVaccine-Derived Multiple HLA Binding Epitopes Predict ReportedExperimental Clinical Response Rates

A correlation between the ≥2 PEPI3+ Score of immunotherapy vaccinesdetermined in the Model Population described in Example 8 and thereported Disease Control Rate (DCR, proportion of patients with completeresponses and partial responses and stable disease) determined inclinical trials was determined.

Seventeen clinical trials conducted with peptide- and DNA-based cancerimmunotherapy vaccines that have published Disease Control Rates (DCRs)or objective response rate (ORR) were identified from peer reviewedscientific journals (Table 15). These trials involved 594 patients (5ethnicities) and covered 29 tumor and viral antigens. DCRs weredetermined according to the Response Evaluation Criteria in Solid Tumors(RECIST), which is the current standard for clinical trials, in whichclinical responses are based on changes in maximum cross-sectionaldimensions^(42, 43, 44) In case there was no available DCR data,objective response rate (ORR) data was used, which is also definedaccording to the RECIST guidelines.

Table 16 compares the ≥2 PEPI3+ Score for each vaccine in the ModelPopulation and the published DCR or ORR. A correlation between thepredicted and measured DCR was observed providing further evidence thatnot only the immunogenicity but also the potency of cancer vaccinesdepends on the multiple HLA sequences of individuals (R2=0.76) (FIG. 9).

TABLE 15 Clinical trials selected for Disease Control Rate (DCR)prediction. HLA Assess- Immuno- Pop. Study pop./ restric- Adm. DoseDosing ment time therapy Antigen Sponsor Disease (n) Ethnicity tion form(mg) schedule (weeks) Ref. IMA901 9 TAAs Immatics Renal cell 28 CEU A02i.d. 0.4 8x in 10 12 19 phase I cancer wks IMA901 9 TAAs Immatics Renalcell 68 CEU A02 i.d 0.4 7x in 5 24 19 phase II cancer wks then 10x 3 wksIpilimumab NY-ESO-1 MSKCC Melanoma 19 US no i.v. 0.3 4 x every 24 5 3 3wks 10 HPV-SLP* HPV-16 E6, E7 Leiden VIN 20 CEU no s.c. 0.3 3 x every 129 University 3 wks HPV-SLP* Leiden HPV-related 5 CEU no s.c. 0.3 3 xevery 12 (OR) 10 University cervical 3 wks cancer gp100 - 2 gp100 BMSMelanoma 136 US A*0201 s.c. 1 4 x every 12 ²8 peptides* 3 wks ImmucinMuc-1 VaxilBio Myeloma 15 Israeli no s.c. 0.1 6 x every  12** ²⁹ 2 wksStimuVax Muc-1 Merck NSCLC 80 Canadian no s.c. 1 8x wkly then 12 13, 30every 6 wks VGX-3100 HPV-16&18 Inovio HPV-related 125 US no i.m. 6 0, 4,12 wks 36 ³1 cervical cancer TSPP Thymidylate Siena CRC, NSCLC, 21 CEUno s.c. 0.1 3 x 3 wks 12 32 peptide synthase University Gallbladder 0.2vaccine carc., Breast-, 0.3 Gastric cancer KIF20A-66 KIF20A ChibaMetastatic 29 Japanese A*2402 s.c. 1 2 cycles 1, 12 (OR) 33 peptideTokushukai pancreatic 3 8, 15, 22 vaccine* Hospital cancer days thenevery 2 wks Peptide 3 TAAs Kumamoto HNSCC 37 Japanese A*2402 s.c. 1 8 xwkly then 12 ³4 vaccine* University every 4 wks 7-peptide 7 TAAs KinkiMetastatic 30 Japanese A*2402 s.c. 1 Cycles: 5 x 10 (OR) ³5 cocktailUniversity colorectal wkly then 1 vaccine* cancer wk rest GVX301* hTERTUniversity Prostate 14 Japanese A02 i.d. 0.5 1, 3, 5, 7, 12 25 Genoa andrenal 14, 21, 35, cancer 63 days MAGE-A3 MAGE-A3 Abramson Multiple 26 USno s.c. 0.3 14, 42, 90, 24 ³6 Trojan* Cancer Center myeloma 120, 150days PepCan HPV-16 E6 University of CIN2/3 23 US no i.m. 0.05 4 x 3 wks24 ³7 Arkansas 0.1 0.25 0.5 Melanoma Tyrosinase, University of Melanoma26 US A1, A2 s.c. 0.1 6 cycles: 0,  6 27 peptide gp100 Virginia or A3 7,14, 28, vaccine* 35, 42 days *Montanide ISA51 VG as adjuvant **Diseaseresponse was assessed according to the International Myeloma WorkingGroup response criteria⁴⁵

TABLE 16 The Disease Control Rates (DCRs) and MultiPEPI Scores(predicted DCR) in 17 clinical trials. MultiPEPI Score OverallPercentage of Immunotherapy DCR (Predicted DCR) Agreement IMA901 phase I43% 27% 61% IMA901 phase II 22% 27% 81% Ipilimumab 60% 65% 92% HPV-SLP60% 70% 86% HPV-SLP 62% 70% 89% gp100 - 2 peptides 15% 11% 73% Immucin73% 59% 81% StimuVax  0%  0% 100%  VGX-3100 50% 56% 89% TSPP peptidevaccine 48% 31% 65% KIF20A-66 peptide 26%  7% 27% vaccine Peptidevaccine 27% 10% 37% 7-peptide cocktail 10%  9% 90% vaccine GVX301 29% 7% 24% MAGE-A3 Trojan 35% 10% 29% PepCan 52% 26% 50% Melanoma peptide12%  6% 50% vaccine

Example 13—Breast Cancer Vaccine Design for Large Population andComposition

We used the PEPI3+ Test described above to design peptides for use inbreast cancer vaccines that are effective in a large percentage ofpatients, taking into account the heterogeneities of both tumourantigens and patients' HLAs.

Breast cancer CTAs were identified and ranked based on the overallexpression frequencies of antigens found in breast cancer tumor samplesas reported in peer reviewed publications (Chen et al. MultipleCancer/Testis Antigens Are Preferentially Expressed in Hormone-ReceptorNegative and High-Grade Breast Cancers. Plos One 2011; 6(3): e17876.;Kanojia et al. Sperm-Associated Antigen 9, a Novel Biomarker for EarlyDetection of Breast Cancer. Cancer Epidemiol Biomarkers Prev 2009;18(2):630-639.; Saini et al. A Novel Cancer Testis Antigen, A-KinaseAnchor Protein 4 (AKAP4) Is a Potential Biomarker for Breast Cancer.Plos One 2013; 8(2): e57095).

Based on the ranked expression rate we have selected the most frequentlyexpressed CTA as target antigens for breast cancer vaccine. Theexpression rates of the selected breast cancer specific CTAs areillustrated in FIG. 11 .

To select immunogenic peptides from the target CTAs we used the PEPI3+Test and the Model Population described in Example 8 to identify the 9mer epitopes (PEPI3+s) that are most frequently presented by at least 3HLAs of the individuals in the Model Population. We refer to theseepitopes herein as “bestEPIs”. An illustrative example of the “PEPI3+hotspot” analysis and bestEPI identification is shown in FIG. 10 for thePRAME antigen.

We multiplied the reported expression frequency for each CTA by thefrequency of the PEPI3+ hotspots in the Model Population to identify theT cell epitopes (9 mers) that will induce a cytotoxic T cell responseagainst breast cancer antigens in the highest proportion of individuals(Table 17). We then selected 15 mers encompassing each of the selected 9mers (Table 17). The 15 mers were selected to bind to most HLA class IIalleles of most subjects, using the process described in Example 19below. These 15 mers can induce both CTL and T helper responses in thehighest proportion of subjects.

TABLE 17BestEPI list (9-mers underlined) for selecting breast cancer peptides forvaccine composition. N %: Antigen expression frequency in colorectal cancers; B %:bestEPI frequency, ie. the percentage of individuals with epitopes binding to at least 3HLA class I of subjects in the model population (433 subjects); HLAII**: Percentage ofindividuals having CD4+ T cell specific PEPI4+ within normal donors (n = 400);N % * B %: N % multiplied by B %. SEQ SEQ ID IDBestEPIs and Optimized 15mer NO. NO. Antigen Opt. HLAII** 9mer 15merAntigen N % Opt. 15mer Position B % (CD4) B % * N % 172 195 PIWIL-2 94%FVASINLTLTKWYSR 760 67% 93% 64% 173 196 PIWIL-2 94% RNFYDPTSAMVLQQH 34160% 49% 57% 1 41 AKAP4 85% DQVNIDYLMNRPQNL 161 52% 46% 44% 1 197 AKAP485% VNIDYLMNRPQNLRL 163 52% 57% 44% 174 198 EpCam 84% RTYWIIIELKHKARE140 51% 100%  43% 2 42 AKAP4 85% MMAYSDTTMMSDDID 1 49%  0% 41% 3 43BORIS 71% MFTSSRMSSFNRHMK 263 57% 66% 40% 3 199 BORIS 71%VCMFTSSRMSSFNRH 261 57% 96% 40% 175 200 HIWI 100%  HAFDGTILFLPKRLQ 16139% 83% 39% 4 201 AKAP4 85% SDLQKYALGFQHALS 116 46% 81% 39% 4 44 AKAP485% LQKYALGFQHALSPS 118 46% 88% 39% 24 64 SPAG9 88% GTGKLGFSFVRITAL 113744% 94% 39% 24 202 SPAG9 88% KLGFSFVRITALMVS 1140 44% 100%  39% 5 45SPAG9 88% AQKMSSLLPTMWLGA 962 43% 69% 38% 176 203 PIWIL-2 94%YSRVVFQMPHQEIVD 772 40% 77% 38% 177 204 HIWI 100%  GFTTSILQYENSIML 25137% 86% 37% 178 205 PLU-1 82% LRYRYTLDDLYPMMN 732 45% 84% 37% 179 206TSGA10 70% YSSNAYHMSSTMKPN 653 48% 33% 34% 180 207 TSGA10 70%LQKVQFEKVSALADL 494 46% 97% 32% 181 208 PLU-1 82% NRTSYLHSPFSTGRS 132138% 37% 31% 6 46 SPAG9 88% GNILDSFTVCNSHVL 779 36%  4% 31% 6 209 SPAG988% LDSFTVCNSHVLCIA 782 36%  6% 31% 7 47 BORIS 71% NMAFVTSGELVRHRR 31944% 75% 31% 182 210 ODF-4 63% NSPLPFQWRITHSFR 63 49% 35% 30% 183 211SP17 47% AFAAAYFESLLEKRE 37 65% 100%  30% 184 212 AKAP4 85%DLSFYVNRLSSLVIQ 216 36% 100%  30% 185 213 ODF-4 63% QDGRLLSSTLSLSSN 4147% 75% 29% 186 214 RHOXF-2 60% WEEAYTFEGARYYIN 62 48% 79% 29% 187 215PLU-1 82% EKAMARLQELLTVSE 955 34% 69% 28% 188 216 HIWI 100% RSIAGFVASINEGMT 642 28% 57% 28% 8 48 PRAME 53% LERLAYLHARLRELL 457 52%100%  28% 189 217 RHOXF-2 60% SDYAVHPMSPVGRTS 132 43%  5% 26% 190 218NY-SAR-35 55% MMQMFGLGAISLILV 184 46% 69% 25% 11 51 NY-SAR-35 55%FSSSGTTSFKCFAPF 163 45%  0% 25% 11 219 NY-SAR-35 55% LRHKCCFSSSGTTSF 15745%  1% 25% 9 49 SPAG9 88% SGAVMSERVSGLAGS 16 28%  9% 25% 10 220 BORIS71% RFTQSGTMKIHILQK 406 35% 69% 25% 10 50 BORIS 71% HTRFTQSGTMKIHIL 40435% 80% 25% 191 221 EpCam 84% QTLIYYVDEKAPEFS 246 28% 34% 24% 13 222NY-SAR-35 55% FVLANGHILPNSENA 97 42%  6% 23% 13 53 NY-SAR-35 55%CSGSYFVLANGHIL 91 42% 78% 23% 13 223 NY-SAR-35 55% SSYFVLANGHILPNS 9442% 85% 23% 12 224 MAGE-A9 44% FMFQEALKLKVAELV 102 49% 100%  22% 12 52MAGE-A9 44% QLEFMFQEALKLKVA 99 49% 100%  22% 14 54 PRAME 53%RHSQTLKAMVQAWPF 64 37% 38% 20% 14 225 PRAME 53% HSQTLKAMVQAWPFT 65 37%37% 20% 14 226 PRAME 53% QTLKAMVQAWPFTCL 67 37% 85% 20% 15 55 NY-BR-147% YSCDSRSLFESSAKI 424 39%  0% 18% 16 56 Survivin 66% TAKKVRRAIEQLAAM127 26% 26% 17% 192 227 MAGE-Ail 59% SHSYVLVTSLNLSYD 286 26% 100%  15%192 228 MAGE-Ail 59% TSHSYVLVTSLNLSY 285 26% 100%  15% 17 229 MAGE-A1159% AMDAIFGSLSDEGSG 184 23%  0% 14% 17 230 MAGE-A11 59% ESFSPTAMDAIFGSL178 23%  0% 14% 17 57 MAGE-Ail 59% SPTAMDAIFGSLSDE 181 23%  0% 14% 18 58HOM-TES-85 47% MASFRKLTLSEKVPP 1 29% 51% 13% 19 59 MAGE-A9 44%SSISVYYTLWSQFDE 67 30% 97% 13% 20 231 NY-BR-1 47% KPSAFEPATEMQKSV 58227%  0% 12% 20 60 NY-BR-1 47% PGKPSAFEPATEMQK 580 27%  0% 12% 193 232NY-ESO-1  9% SRLLEFYLAMPFATP 85 52% 98%  5% 194 233 NY-ESO-1  9%FYLAMPFATPMEAEL 90 51% 96%  5%

Then we designed thirty-one 30 mer peptides (Table 18a). The 30 mers mayeach consist of two optimized 15 mer fragments, generally from differentfrequent CTAs, arranged end to end, each fragment comprising one of the9 mers (BestEPIs) from Table 17. Nine of these 30 mer peptides wereselected for a panel of peptides, referred to as PolyPEPI915 (Table18b). Expression frequencies for the 10 CTAs targeted by PolyPEPI915,singly and in combination, are shown in FIG. 11 .

TABLE 18a 30mer breast cancer vaccine peptides HLAI* HLAII** SEQIDTREOSID Source Antigen Peptide (30mer) (CD8) (CD4) 81 BCV900-2-1 AKAP4LQKYALGFQHALSPSMMAYSDTTMMSDDID 69% 88% 82 BCV900-2-2 BORIS/AKAP4VCMFTSSRMSSFNRHVNIDYLMNRPQNLRL 76% 97% 83 BCV900-2-3 BORISNMAFVTSGELVRHRRHTRFTQSGTMKIHIL 57% 92% 84 BCV900-2-4 SPAG9LDSFTVCNSHVLCIAKLGFSFVRITALMVS 58% 100%  85 BCV900-2-5 SPAG9/NY-SAR-35AQKMSSLLPTMWLGAMMQMFGLGAISLILV 66% 83% 86 BCV900-2-6 PRAMELERLAYLHARLRELLQTLKAMVQAWPFTCL 71% 100%  87 BCV900-2-7 NY-SAR-35SSYFVLANGHILPNSLRHKCCFSSSGTTSF 64% 85% 88 BCV900-2-8 Survivin/MAGE-A9TAKKVRRAIEQLAAMQLEFMFQEALKLKVA 58% 100%  89 BCV900-2-9 MAGE-A11/NY-BR-1TSHSYVLVTSLNLSYYSCDSRSLFESSAKI 65% 100%  90 BCV900-3-1 SPAG9/BORISLDSFTVCNSHVLCIAVCMFTSSRMSSFNRH 65% 96% 91 BCV900-3-2 NY-SAR-35/PRAMELRHKCCFSSSGTTSFQTLKAMVQAWPFTCL 59% 85% 92 BCV900-3-3 NY-BR-1/SURVIVINYSCDSRSLFESSAKITAKKVRRAIEQLAAM 55% 26% 93 BCV900-3-4 AKAP-4/BORISMMAYSDTTMMSDDIDHTRFTQSGTMKIHIL 72% 80% 94 BCV900-3-5 SPAG9/AKAP-4AQKMSSLLPTMWLGALQKYALGFQHALSPS 64% 92% 95 BCV900-3-6 MAGE-A11/BORISTSHSYVLVTSLNLSYNMAFVTSGELVRHRR 61% 100%  96 BCV900-3-7 NY-SAR-35/AKAP-4MMQMFGLGAISLILVVNIDYLMNRPQNLRL 71% 84% 97 BCV900-3-8 NY-SAR-35/SPAG-9SSYFVLANGHILPNSKLGFSFVRITALMVS 65% 100%  98 BCV900-3-9 PRAME/MAGE-A9LERLAYLHARLRELLQLEFMFQEALKLKVA 73% 100%  99 BCV900-4-1 SPAG9/AKAP4GNILDSFTVCNSHVLLQKYALGFQHALSPS 53% 88% 100 BCV900-4-2 BORIS/NY-SAR-35NMAFVTSGELVRHRRFSSSGTTSFKCFAPF 65% 75% 101 BCV900-4-5 SPAG9/BORISAQKMSSLLPTMWLGAMFTSSRMSSFNRHMK 72% 87% 102 BCV900-4-6 MAGE-A11/PRAMETSHSYVLVTSLNLSYHSQTLKAMVQAWPFT 60% 100%  103 BCV900-5-6 HomTes85/MageA11MASFRKLTLSEKVPPSPTAMDAIFGSLSDE 45% 51% 104 BCV900-5-7 AKAP4/PRAMEDQVNIDYLMNRPQNLRHSQTLKAMVQAWPF 64% 67% 105 BCV900-5-8 NYSAR/SPAG9CSGSSYFVLANGHILSGAVMSERVSGLAGS 46% 78% 106 BCV900-S-2 AKAP-4/MAGE-A9DLSFYVNRLSSLVIQSSISVYYTLWSQFDE 60% 100%  107 BCV900-S-4 SPAG9/NY-ESO-1SGAVMSERVSGLAGSSRLLEFYLAMPFATP 59% 98% 108 BCV900-S-6HOM-TES-85/MAGE-A11 MASFRKLTLSEKVPPESFSPTAMDAIFGSL 46% 51% 109BCV900-S-7 NY-ESO-1/NY-BR-1 FYLAMPFATPMEAELKPSAFEPATEMQKSV 60% 96% 110BCV900-T-27 MAGE-A11/PRAME AMDAIFGSLSDEGSGHSQTLKAMVQAWPFT 54% 37% 111BCV900-T-28 NY-SAR-35/SPAG9 FVLANGHILPNSENAGTGKLGFSFVRITAL 61% 94% 435BCV900-6-1 TSGA10/PIWIL-2 YSSNAYHMSSTMKPNFVASINLTLTKWYSR 80% 95% 436BCV900-6-2 PIWIL-2/AKAP4 RNFYDPTSAMVLQQHMMAYSDTTMMSDDID 88% 49% 437BCV900-6-3 PLU-1/RHOXF-2 LRYRYTLDDLYPMMNSDYAVHPMSPVGRTS 67% 85% 438BCV900-6-4 SPAG9/EpCam SGAVMSERVSGLAGSRTYWIIIELKHKARE 60% 100%  439BCV900-6-5 AKAP4/PLU-1 DLSFYVNRLSSLVIQNRTSYLHSPFSTGRS 66% 100%  440BCV900-6-6 AKAP4/HIWI VNIDYLMNRPQNLRLHAFDGTILFLPKRLQ 70% 94% 441BCV900-6-7 AKAP4/PLU-1 SDLQKYALGFQHALSEKAMARLQELLTVSE 56% 92% 442BCV900-6-8 HIWI/ODF-4 GFTTSILQYENSIMLQDGRLLSSTLSLSSN 61% 94% 443BCV900-6-9 PIWIL-2/BORIS YSRVVFQMPHQEIVDNMAFVTSGELVRHRR 61% 85% 444BCV900-6-10 SP17/BORIS AFAAAYFESLLEKREMFTSSRMSSFNRHMK 82% 100%  445BCV900-6-11 ODF-4/HIWI NSPLPFQWRITHSFRRSIAGFVASINEGMT 60% 69% 446BCV900-6-12 NY-SAR-35/RHOXF-2 SSYFVLANGHILPNSWEEAYTFEGARYYIN 74% 93% 447BCV900-6-13 TSGA10/PRANIE LQKVQFEKVSALADLLERLAYLHARLRELL 68% 100%  448BCV900-6-14 MAGE-A11/MAGE-A9 SHSYVLVTSLNLSYDFMFQEALKLKVAELV 65% 100% 449 BCV900-6-15 BORIS/EpCam RFTQSGTMKIHILQKQTLIYYVDEKAPEFS 53% 80%

TABLE 18b Selected Breast Cancer Vaccine peptides for PolyPEPI915panel/composition HLAI* HLAII** SEQID TREOSID Source AntigenPeptide (30mer) (CD8) (CD4) 99 BCV900-4-1 SPAG9/AKAP4GNILDSFTVCNSHVLLQKYALGFQHALSPS 53% 75% 100 BCV900-4-2 BORIS/NY-SAR-35NMAFVTSGELVRHRRFSSSGTTSFKCFAPF 65% 46% 92 BCV900-3-3 NY-BR-1/SURVIVINYSCDSRSLFESSAKITAKKVRRAIEQLAAM 55% 11% 93 BCV900-3-4 AKAP-4/BORISMMAYSDTTMMSDDIDHTRFTQSGTMKIHIL 72% 45% 101 BCV900-4-5 SPAG9/BORISAQKMSSLLPTMWLGAMFTSSRMSSFNRHMK 72% 50% 103 BCV900-5-6 HomTes85/MageA11MASFRKLTLSEKVPPSPTAMDAIFGSLSDE 45% 16% 104 BCV900-5-7 AKAP4/PRAMEDQVNIDYLMNRPQNLRHSQTLKAMVQAWPF 64% 33% 105 BCV900-5-8 NYSAR/SPAG9CSGSSYFVLANGHILSGAVMSERVSGLAGS 46% 48% 98 BCV900-3-9 PRAME/MAGE-A9LERLAYLHARLRELLQLEFMFQEALKLKVA 73% 100% PolyPEPI915 (9 peptide together)  96% 100%  *Percentage of individualshaving CD8+ T cell specific PEPI3+ within the HLA class I ModelPopulation (n = 433). **Percentage of individuals having CD4+ T cellspecific PEPI4+ within the normal donors (n = 400).

Characterization of PolyPEPI915

Tumor heterogeneity can be addressed by including peptide sequences thattarget multiple CTAs in a vaccine or immunotherapy regime. ThePolyPEPI915 composition targets 10 different CTAs. Based on the antigenexpression rates for these 10 CTAs, we modelled the predicted averagenumber of expressed antigens (AG50) and the minimum number of expressedantigens with 95% likelihood (AG95) in the cancer cells. 95% ofindividuals expressed minimum 4 of the 10 target antigens (AG95=4) asshown by the antigen expression curve in FIGS. 12A-B.

The AG values described above characterize a vaccine independently fromthe target patient population. They can be used to predict thelikelihood that a specific cancer (e.g. breast cancer) expressesantigens targeted by a specific vaccine or immunotherapy composition. AGvalues are based on known tumor heterogeneity, but do not take HLAheterogeneity into account.

HLA heterogeneity of a certain population can be characterised from theviewpoint of an immunotherapy or vaccine composition by the number ofantigens representing PEPI3+. These are the vaccine-specific CTAantigens for which ≥1 PEPI3+ is predicted, referred to herein as the“AP”. The average number of antigens with PEPI3+(AP50) shows how thevaccine can induce immune response against the antigens targeted by thecomposition (breast cancer vaccine specific immune response). ThePolyPEPI915 composition can induce immune response against an average of5.3 vaccine antigens (AP50=5.30) and 95% of the Model Population caninduce immune response against at least one vaccine antigen(AP95=1)(FIGS. 13A-B).

Vaccines can be further characterized by AGP values that refers toantigens with PEPIs”. This parameter is the combination of the previoustwo parameters: (1) AG is depending on the antigen expressionfrequencies in the specific tumor type but not on the HLA genotype ofindividuals in the population, and (2) AP is depending on the HLAgenotype of individuals in a population without taking account theexpression frequencies of the antigen. The AGP is depending on both, theexpression frequencies of vaccine antigens in the disease and the HLAgenotype of individuals in a population.

Combining the data of AG of breast cancer and AP in the Model Populationwe determined the AGP value of PolyPEPI915 that represents theprobability distribution of vaccine antigens that are induce immuneresponses against antigens expressed in breast tumors. For PolyPEPI915,the AGP50 value in the Model Population is 3.37. The AGP92=1, means that92% of the subjects in the Model Population induce immune responsesagainst at least one expressed vaccine antigen (FIGS. 14A-B).

Example 14—Patient Selection Using Companion Diagnostic for BreastCancer Vaccine

The likelihood that a specific patient will have an immune response or aclinical response to treatment with one or more cancer vaccine peptides,for example as described above, can be determined based on (i) theidentification of PEPI3+ within the vaccine peptide(s) (9 mer epitopescapable of binding at least three HLA of the patient); and/or (ii) adetermination of target antigen expression in cancer cells of thepatient, for example as measured in a tumour biopsy. Ideally bothparameters are determined and the optimal combination of vaccinepeptides is selected for use in treatment of the patient. However,PEPI3+ analysis alone may be used if a determination of the expressedtumour antigens, for example by biopsy, is not possible, not advised, orunreliable due to biopsy error (i.e. biopsy tissue samples taken from asmall portion of the tumor or metastasised tumors do not represent thecomplete repertoire of CTAs expressed in the patient).

Example 15—Comparison of PolyPEPI915 with Competing Breast CancerVaccines

We used the in silico clinical trial model described in above to predictthe immune response rates of competing breast cancer vaccines thatinvestigated in clinical trials (Table 19). The immune response rate ofthese products were between 3% and 91%.

The single peptide vaccines were immunogenic in 3%-23% of individuals.In comparison, peptides having an amino acid sequence selected from SEQID NOs: 81-111 were immunogenic in from 44% to 73% of individuals in thesame cohorts. This result represents substantial improvement inimmunogenicity of each peptide in PolyPEPI915.

Competing combination peptide products immune response rates werebetween 10-62%. The invented PolyPEPI915 combination product were 96% inthe Model Population and 93% in a breast cancer patient populationrepresenting improvement in immunogenicity.

TABLE 19 Predicted immune response rates of competing breast cancervaccines Predicted immune response rates* 433 normal 90 patients Targetdonors (Model with breast Breast Cancer Vaccines Sponsors antigensPopulation) cancer DPX0907 Multipeptide ImmunoVaccine 7 58% 62% Tech.Multipeptide vaccine University of 5 22% 31% VirginiaAd-sig-hMUC-1/ecdCD40L Singapore CRI 1 91% 80% NY-ESO-1 IDC-G305 ImmuneDesign 1 84% 84% Corp. 6 HER2 peptide pulsed DC University 1 29% 36%Pennsylvania HER-2 B Cell peptide Ohio State 1 18% 23% UniversityHER-2/neu ID protein University 1 10% 11% Washington NeuVax peptideGalena Biopharma 1  6%  3% StimuVax ®(L-BLP25) peptide EMD Serono 1  6% 8% PolyPEPI915 Treos Bio 10 96% 93% *Proportion of subjects with ≥1PEPI3+

Another improvement of using the PolyPEPI915 vaccine is the lower chanceof tumor escape. Each 30 mer peptide in PolyPEPI915 targets 2 tumorantigens. CTLs against more tumor antigens are more effective againstheterologous tumor cells that CTLs against a single tumor antigen.

Another improvement is that PolyPEPI915 vaccine that individuals wholikely respond to vaccination can be identified based on their HLAgenotype (sequence) and optionally antigen expression in their tumorusing the methods described here. Pharmaceutical compositions withPolyPEPI vaccines will not be administered to individuals whose HLAcannot present any PEPI3 from the vaccines. During clinical trialscorrelation will be made between the mAGP or number of AGP in thePolyPEPI915 regimen and the duration of individual's responses. Avaccine combination with >1 AGP is most likely required to destroyheterologous tumor cells. Pharmaceutical compositions with PolyPEPIvaccines will not be administered to individuals whose HLA cannotpresent any PEPI3 from the vaccines.

Example 16 Colorectal Cancer Vaccine Design and Composition

We show another example for colorectal vaccine composition using thesame design method demonstrated above. We used the PEPI3+ Test describedabove to design peptides for use in colorectal cancer vaccines that areeffective in a large percentage of patients, taking into account theheterogeneities of both tumour antigens and patient HLAs.

Colorectal cancer CTAs were identified and ranked based on the overallexpression frequencies of antigens found in breast cancer tumor samplesas reported in peer reviewed publications (FIG. 15 ) (Choi J, Chang H.The expression of MAGE and SSX, and correlation of COX2, VEGF, andsurvivin in colorectal cancer. Anticancer Res 2012. 32(2):559-564.;Goossens-Beumer I J, Zeestraten E C, Benard A, Christen T, Reimers M S,Keijzer R, Sier C F, Liefers G J, Morreau H, Putter H, Vahrmeijer A L,van de Velde C J, Kuppen P J. Clinical prognostic value of combinedanalysis of Aldh1, Survivin, and EpCAM expression in colorectal cancer.Br J Cancer 2014. 110(12):2935-2944.; Li M, Yuan Y H, Han Y, Liu Y X,Yan L, Wang Y, Gu J. Expression profile of cancer-testis genes in 121human colorectal cancer tissue and adjacent normal tissue. ClinicalCancer Res 2005. 11(5):1809-1814).

Based on the ranked expression rate we have selected the most frequentlyexpressed CTA as target antigens for the colorectal cancer vaccine. Theexpression rates of the selected breast cancer specific CTAs areillustrated in FIG. 15 .

To select immunogenic peptides from the most frequently expressedcolorectal cancer CTAs we used the PEPI3+ Test and the Model Populationdescribed in Example 8 to identify the “bestEPIs”.

We multiplied the reported expression frequency for each CTA (N %) bythe frequency of the PEPI3+ hotspots in the Model Population (B %) toidentify the T cell epitopes (9 mers) that will induce an immuneresponse against colorectal cancer antigens in the highest proportion ofindividuals (Table 20). We then selected 15 mers encompassing each ofthe selected 9 mers (Table 20). The 15 mers were selected to bind tomost HLA class II alleles of most subjects, using the process describedin Example 19 below. These 15 mers can induce both CTL and T helperresponses in the highest proportion of subjects.

TABLE 20BestEPI list (9-mers underlined) for selecting colorectal cancer peptides forvaccine composition. N %: Antigen expression frequency in colorectal cancers; B %:bestEPI frequency, ie. the percentage of individuals with epitopes binding to at least 3HLA class I of subjects in the model population (433 subjects); HLAII**: Percentage ofindividuals having CD4+ T cell specific PEPI4+ within normal donors (n = 400);N % * B %: N % multiplied by B %. SEQ SEQ ID IDBestEPIs and Optimized 15 mer NO. NO. Antigen Opt. HLAII** 9mer 15merAntigen N % Opt. 15mer  Position B % (CD4) B % * N % 234 251 TSP50 89%VCSMEGTWYLVGLVS 315 58% 72% 52% 21 252 TSP50 89% GFSYEQDPTLRDPEA 105 51% 0% 45% 21 61 TSP50 89% RSCGFSYEQDPTLRD 102 51%  0% 45% 21 253 TSP50 89%YRSCGFSYEQDPTLR 101 51%  0% 45% 22 62 EpCAM 88% VRTYWIIIELKHKAR 139 51%100%  45% 235 254 EpCAM 88% LLAAATATFAAAQEE 12 39% 28% 34% 24 255 SPAG974% KLGFSFVRITALMVS 1140 44% 100%  33% 23 63 TSP50 89% PSTTMETQFPVSEGK83 36%  0% 32% 24 64 SPAG9 74% GTGKLGFSFVRITAL 1137 44% 94% 32% 23 256TSP50 89% LPSTTMETQFPVSEG 82 36%  0% 32% 25 65 SPAG9 74% AQKMSSLLPTMWLGA962 43% 69% 32% 26 66 CAGE1 74% LASKMHSLLALMVGL 613 42% 99% 31% 27 67FBXO39 39% KFMNPYNAVLTKKFQ 95 78% 43% 30% 28 68 CAGE1 74%PKSMTMMPALFKENR 759 37% 87% 27% 238 257 SPAG9 74% LDSFTVCNSHVLCIA 78236%  6% 27% 236 258 SPAG9 74% GNILDSFTVCNSHVL 779 36%  4% 26% 29 69EpCAM 88% YVDEKAPEFSMQGLK 251 28%  0% 25% 29 259 EpCAM 88%QTLIYYVDEKAPEFS 246 28% 34% 25% 30 70 FBXO39 39% FKKTMSTFHNLVSLN 216 58%92% 23% 31 71 Survivin 86% TAKKVRRAIEQLAAM 127 26% 26% 22% 237 260 TSP5089% SRTLLLALPLPLSLL 368 24% 100%  21% 32 72 SPAG9 74% SGAVMSERVSGLAGS 1628%  9% 21% 238 260 TSP50 89% SRTLLLALPLPLSLL 368 23% 100%  20% 34 74FBXO39 39% KVNFFFERIMKYERL 284 46% 100%  18% 33 73 TSP50 89%SRYRAQRFWSWVGQA 190 20% 88% 18% 239 261 LEMD1 56% FIIVVFVYLTVENKS 16430% 97% 17% 240 66 CAGE1 74% LASKMHSLLALMVGL 613 22% 99% 16% 241 262FBXO39 39% RNSIRSSFISSLSFF 142 40% 100%  16% 242 263 CAGE1 74%NIENYSTNALIQPVD 97 21% 14% 16% 243 264 Survivin 86% MGAPTLPPAWQPFLK 117%  0% 15% 244 265 CAGE1 74% RQFETVCKFHWVEAF 119 18% 45% 13% 35 75Survivin 86% KDHRISTFKNWPFLE 15 15% 83% 13% 36 266 MAGE-A8 44%PEEAIWEALSVMGLY 220 20% 78%  9% 36 76 MAGE-A8 44% SRAPEEAIWEALSVM 21720%  6%  9% 37 77 MAGE-A8 44% DEKVAELVRFLLRKY 113 18% 95%  8% 37 267MAGE-A8 44% EKVAELVRFLLRKYQ 114 18% 99%  8% 38 268 MAGE-A6 28%KLLTQYFVQENYLEY 244 27% 98%  8% 38 78 MAGE-A6 28% QYFVQENYLEYRQVP 24827% 93%  8% 40 80 MAGE-A6 28% IGHVYIFATCLGLSY 172 25% 82%  7% 39 79MAGE-A8 44% EFLWGPRALAETSYV 273 16% 44%  7% 245 269 MAGE-A3 23%IGHLYIFATCLGLSY 172 28% 85%  6% 246 270 MAGE-A3 23% KLLTQHFVQENYLEY 24427% 77%  6% 247 271 MAGE-A8 44% ASSSSTLIMGTLEEV 39 14% 19%  6% 248 269MAGE-A3 23% IGHLYIFATCLGLSY 172 25% 85%  6% 249 264 Survivin 86%MGAPTLPPAWQPFLK 1  5%  0%  4% 250 75 Survivin 86% KDHRISTFKNWPFLE 15  4%83%  3%

Then we designed thirty-one 30 mer peptides (Table 21a). The 30 merseach consist of two optimized 15 mer fragments, generally from differentfrequent CTAs, each 30 mer generally containing at least one highfrequency HLA class-II binding PEPI. The 15 mer fragments are arrangedend to end, and each comprises one of the 9 mers (BestEPIs) from Table20 as described above. Nine of these 30 mer peptides were selected for apanel of peptide vaccines, referred to as PolyPEPI1015 (Table 21b).Expression frequencies for the 8 CTAs targeted by PolyPEPI1015, singlyand in combination, are shown in FIG. 15 .

TABLE 21a 30mer colorectal cancer vaccine peptides SEQ HLAI* HLAII** IDTREOSID Source Antigen Peptide (30mer) (CD8) (CD4) 112 CCV1000-1-1 TSP50VCSMEGTWYLVGLVSYRSCGFSYEQDPTLR 71% 72% 113 CCV1000-1-2 EpCAM/TSP50VRTYWIIIELKHKARLPSTTMETQFPVSEG 62% 100%  114 CCV1000-1-4 SurvivinTAKKVRRAIEQLAAMMGAPTLPPAWQPFLK 39% 26% 115 CCV1000-1-5 CAGE1LASKMHSLLALMVGLPKSMTMMPALFKENR 68% 99% 116 CCV1000-1-6 Spag9KLGFSFVRITALMVSLDSFTVCNSHVLCIA 58% 100%  117 CCV1000-1-7 FBXO39KFMNPYNAVLTKKFQFKKTMSTFHNLVSLN 91% 92% 118 CCV1000-1-8 Spag9/FBXO39AQKMSSLLPTMWLGAKVNFFFERIMKYERL 75% 100%  119 CCV1000-1-9Survivin/Mage-A8 KDHRISTFKNWPFLEPEEAIWEALSVMGLY 39% 93% 120 CCV1000-2-1TSP50 YRSCGFSYEQDPTLRVCSMEGTWYLVGLVS 71% 72% 121 CCV1000-2-2EpCAM/Survivin VRTYWIIIELKHKARTAKKVRRAIEQLAAM 57% 100%  122 CCV1000-2-4TSP50/Spag9 LPSTTMETQFPVSEGKLGFSFVRITALMVS 61% 100%  123 CCV1000-2-5Survivin/Mage-A8 MGAPTLPPAWQPFLKPEEAIWEALSVMGLY 40% 78% 124 CCV1000-2-6CAGE1/Survivin LASKMHSLLALMVGLKDHRISTFKNWPFLE 58% 99% 125 CCV1000-2-7CAGE1/Spag9 PKSMTMMPALFKENRLDSFTVCNSHVLCIA 61% 87% 126 CCV1000-2-8FBXO39 KFMNPYNAVLTKKFQKVNFFFERIMKYERL 90% 100%  127 CCV1000-2-9Spag9/FBXO39 AQKMSSLLPTMWLGAFKKTMSTFHNLVSLN 67% 92% 128 CCV1000-3-1TSP50 GFSYEQDPTLRDPEAVCSMEGTWYLVGLVS 71% 72% 129 CCV1000-3-7 CAGE1/Spag9PKSMTMMPALFKENRGNILDSFTVCNSHVL 61% 87% 130 CCV1000-5-1 TSP50PSTTMETQFPVSEGKSRYRAQRFWSWVGQA 53% 88% 131 CCV1000-5-3 EpCAM/Mage-A8YVDEKAPEFSMQGLKDEKVAELVRFLLRKY 43% 95% 132 CCV1000-5-4 TSP50/Spag9RSCGFSYEQDPTLRDGTGKLGFSFVRITAL 67% 94% 133 CCV1000-5-5 Mage-A8/Mage-A6SRAPEEAIWEALSVMQYFVQENYLEYRQVP 45% 94% 134 CCV1000-5-7 CAGE1/Spag9PKSMTMMPALFKENRSGAVMSERVSGLAGS 57% 87% 135 CCV1000-S-1 SPAG9/FBXO39SGAVMSERVSGLAGSRNSIRSSFISSLSFF 64% 100%  136 CCV1000-S-2 CAGE1/MAGE-A8NIENYSTNALIQPVDEKVAELVRFLLRKYQ 28% 99% 137 CCV1000-S-3 CAGE1/MAGE-A6RQFETVCKFHWVEAFKLLTQYFVQENYLEY 46% 98% 138 CCV1000-S-5 MAGE-A8/MAGE-A3EFLWGPRALAETSYVKLLTQHFVQENYLEY 39% 91% 139 CCV1000-S-6 MAGE-A8/EpCAMASSSSTLIMGTLEEVQTLIYYVDEKAPEFS 41% 41% 140 CCV1000-S-7 TSP50/MAGE-A3SRTLLLALPLPLSLLIGHLYIFATCLGLSY 60% 100%  141 CCV1000-S-9 LEMD1/MAGE-A6FIIVVFVYLTVENKSIGHVYIFATCLGLSY 51% 99% 142 CCV1000-S-17 EPCAMLLAAATATFAAAQEEQTLIYYVDEKAPEFS 52% 54% *Percentage of individuals havingCD8+ T cell specific PEPI3+ within the Model Population (n = 433).**Percentage of individuals having CD4+ T cell specific PEPI4+ withinnormal donors (n = 400).

TABLE 21bSelected Colorectal Cancer Vaccine peptides for PolyPEPI1015 compositionHLAI* HLAII** SEQID TREOSID Source Antigen Peptide (30mer) (CD8) (CD4)130 CCV1000-5-1 TSP50 PSTTMETQFPVSEGKSRYRAQRFWSWVGQA 53% 53% 121CCV1000-2-2 EpCAM/Survivin VRTYWIIIELKHKARTAKKVRRAIEQLAAM 57% 98% 131CCV1000-5-3 EpCAM/Mage-A8 YVDEKAPEFSMQGLKDEKVAELVRFLLRKY 43% 72% 132CCV1000-5-4 TSP50/Spag9 RSCGFSYEQDPTLRDGTGKLGFSFVRITAL 67% 82% 133CCV1000-5-5 Mage-A8/Mage-A6 SRAPEEAIWEALSVMQYFVQENYLEYRQVP 45% 76% 124CCV1000-2-6 CAGE1/Survivin LASKMHSLLALMVGLKDHRISTFKNWPFLE 58% 95% 134CCV1000-5-7 CAGE1/Spag9 PKSMTMMPALFKENRSGAVMSERVSGLAGS 57% 57% 126CCV1000-2-8 FBXO39 KFMNPYNAVLTKKFQKVNFFFERIMKYERL 90% 98% 127CCV1000-2-9 Spag9/FBXO39 AQKMSSLLPTMWLGAFKKTMSTFHNLVSLN 67% 66%PolyPEPI1015 (9 peptide together) 100%  99% *Percentage of individualshaving CD8+ T cell specific PEPI3+ within the Model Population (n= 433). **Percentage of individuals having CD4+ T cell specificPEPI4+ within normal donors (n = 400).

Characterization of PolyPEPI1015 Colorectal Cancer Vaccine

Tumor heterogeneity: The PolyPEPI1015 composition targets 8 differentCTAs (FIG. 15 ). Based on the antigen expression rates for these 8 CTAs,AG50=5.22 and AG95=3 FIGS. 16A-B.

Patient heterogeneity: the AP50=4.73 and AP95=2 (AP95=2) (FIGS. 17A-B).Both tumor and

patient heterogeneity: AGP50=3.16 and AGP95=1 (Model Population) (FIGS.18A-B).

Example 17—Comparison of Colorectal Cancer Vaccine Peptides withCompeting Colorectal Cancer Vaccines

We used the in silico clinical trial model described above to determineT cell responder rate of state of art and currently developed CRCpeptide vaccines and compared to and compared to that of polyPEPI1015(Table 22). Our PEPI3+ test demonstrate that competing vaccines caninduce immune responses against one tumor antigen in a fraction ofsubjects (2%-77%). However, the multi-antigen (multi-PEPI) responsedetermination for the 2 competitor multi-antigen vaccines resulted in noor 2% responders. *% of responders are the ratio of subjects from theModel population with 1≥PEPI3+ for HLAI (CD8+ T cell responses) in caseof 1, or for 2, 3, 4 or 5 antigens of the vaccine compositions. Sincemulti-PEPI responses correlate with clinical responses induced by tumorvaccines, it is unlikely that any of the competing vaccines willdemonstrate clinical benefit in 98% of patients. In contrast, wepredicted multi-PEPI responses in 95% of subjects suggesting thelikelihood for clinical benefit in the majority of patients.

TABLE 22 Predicted immune response rates of polyPEPI1015 and competingcolorectal cancer vaccines % of CD8+ T cell responders in 433 subjects*Vaccine Colorectal Cancer antigens % responders against multiple AgsVaccines Sponsor (Ags) 1 Ag 2 Ags 3 Ags 4 Ags 5 Ags Stimuvax ®(L-BLP25)Johannes Gutenberg 1 6% — — — — Peptide Vaccine University Mainz WT1Multipeptide Shinshu University, 1 79%  — — — — Vaccine JapanMultiepitope Peptide Kinki University 7 5% 2% 0% 0% 0% Cocktail Vaccinep53 Synthetic Long Leiden University 1 77%  — — — — Peptide VaccineMedical Center HER-2 B Cell Peptide Ohio State University 1 18%  — — — —Vaccine Comprehensive Cancer Center NY-ESO-1 peptide JonssonComprehensive 1 0% — — — — pulsed dendritic Cancer Center cell vaccineOCV-C02 Otsuka Pharmaceutical 2 2% 0% — — — Co., Ltd. PolyPEPI1015 TreosBio 8 100%  95%  87%  70%  54% 

Example 18 Ovarian Cancer Vaccine Design and Composition

We used the PEPI3+ Test to design peptides for use in ovarian cancervaccines using essentially the same design method described in Examples13 and 16 above.

We multiplied the reported expression frequency for CTAs associated withovarian cancer (N %) by the frequency of the PEPI3+ hotspots in theModel Population (B %) to identify the T cell epitopes (9 mers) thatwill induce an immune response against ovarian cancer antigens in thehighest proportion of individuals (Table 23). We then selected 15 mersencompassing each of the selected 9 mers (Table 23). The 15 mers wereselected to bind to most HLA class II alleles of most subjects, usingthe process described in Example 20 below.

TABLE 23BestEPI list (9-mers underlined) for selecting ovarian cancer peptides for vaccinecomposition. N %: Antigen expression frequency in colorectal cancers; B %: bestEPI frequency, ie. the percentage of individuals with epitopes binding to at least 3HLA  class I of subjects in the model population (433 subjects); HLAII**:Percentage of individuals having CD4+ T cell specificPEPI4+ within normal donors (n = 400); N % * B %: N % multiplied by B %.SEQ SEQ ID ID BestEPIs and Optimized 15mer NO. NO. Antigen Opt. HLAII**9mer 15mer Antigen N % Opt. 15mer Position B % (CD4) B % * N % 272 302PIWIL-4 90% QGMMMSIATKIAMQM 585 79% 72% 71% 273 303 PIWIL-4 90%KAKAFDGAILFLSQK 153 62% 80% 56% 274 304 WT1 63% SSGQARMFPNAPYLP 121 78% 0% 49% 275 305 EpCam 92% RTYWIIIELKHKARE 140 51% 100%  47% 276 306BORIS 82% MFTSSRMSSFNRHMK 263 57% 66% 46% 277 307 AKAP4 88%QVNIDYLMNRPQNLR 162 52% 46% 46% 278 308 OY-TES-1 65% STPMIMENIQELIRS 27767% 82% 43% 279 309 AKAP4 88% MMAYSDTTMMSDDID 1 49%  0% 43% 280 310 SP1765% AFAAAYFESLLEKRE 37 65% 100%  42% 281 311 PIWIL-4 90% RAIQQYVDPDVQLVM534 46%  5% 42% 282 312 PIWIL-2 61% GFVASINLTLTKWYS 759 67% 93% 41% 283313 AKAP4 88% DLQKYALGFQHALSP 117 46% 82% 40% 284 314 PIWIL-3 88%GYVTSVLQYENSITL 266 44% 54% 39% 285 315 SPAG9 90% VREEAQKMSSLLPTM 95843%  1% 39% 286 316 PIWIL-3 88% MSLKGHLQSVTAPMG 523 42% 17% 37% 287 317PIWIL-3 88% QKSIAGFVASTNAEL 663 42% 37% 37% 288 318 PIWIL-2 61%RNFYDPTSAMVLQQH 341 60% 49% 37% 289 319 BORIS 82% NMAFVTSGELVRHRR 31944% 75% 36% 290 320 AKAP4 88% LSFYVNRLSSLVIQM 217 36% 100%  31% 291 321PRAME 59% LERLAYLHARLRELL 457 52% 100%  30% 292 322 BORIS 82%RFTQSGTMKIHILQK 406 35% 69% 29% 293 323 HIWI 68% HAFDGTILFLPKRLQ 161 39%83% 27% 294 324 EpCam 92% YVDEKAPEFSMQGLK 251 28%  0% 26% 295 325 SPAG990% SGAVMSERVSGLAGS 16 28%  9% 25% 296 326 HIWI 68% GFTTSILQYENSIML 25137% 86% 25% 297 327 PIWIL-2 61% YSRVVFQMPHQEIVD 772 40% 77% 24% 298 328PRAME 59% RHSQTLKAMVQAWPF 64 37% 38% 22% 299 329 Survivin 84%AKKVRRAIEQLAAMD 128 26% 25% 22% 300 330 BORIS 82% ERSDEIVLTVSNSNV 21025%  2% 21% 301 331 WT1 63% RTPYSSDNLYQMTSQ 218 32%  0% 20%

Then we designed 15 30 mer peptides (Table 24).

TABLE 24 30mer ovarian cancer vaccine peptides HLAI* HLAII** SEQIDTREOSID Source Antigen Peptide (30mer) (CD8) (CD4) 332 OC1212-01OY-TES-1/PIWIL-4 STPMIMENIQELIRSQGMHMSIATKIAMQM 94% 98% 333 OC1212-02PIWIL-2/PIWIL-4 RNFYDPTSAMVLQQHKAKAFDGAILFLSQK 89% 90% 334 OC1212-03BORIS/AKAP4 NMAFVTSGELVRHRRMMAYSDTTMMSDDID 68% 75% 335 OC1212-04 WT1/WT1SSGQARMFPNAPYLPRTPYSSDNLYQMTSQ 84%  0% 336 OC1212-05 BORIS/HIWIMFTSSRMSSFNRHMKHAFDGTILFLPKRLQ 67% 94% 337 OC1212-06 PIWIL-2/EpCamYSRVVFQMPHQEIVDRTYWIIIELKHKARE 67% 100%  338 OC1212-07 AKAP4/PIWIL-4LSFYVNRLSSLVIQMRAIQQYVDPDVQLVM 71% 100%  339 OC1212-08 AKAP4/SP17QVNIDYLMNRPQNLRAFAAAYFESLLEKRE 78% 100%  340 OC1212-09 PIWIL-3/PIWIL-3GYVTSVLQYENSITLQKSIAGFVASTNAEL 64% 65% 341 OC1212-10 SPAG9/BORISVREEAQKMSSLLPTMRFTQSGTMKIHILQK 62% 69% 342 OC1212-11 PIWIL-2/EpCamGFVASINLTLTKWYSYVDEKAPEFSMQGLK 74% 93% 343 OC1212-12 PIWIL-3/SPAG9MSLKGHLQSVTAPMGSGAVMSERVSGLAGS 52% 19% 344 OC1212-13 AKAP4/PRAMEDLQKYALGFQHALSPLERLAYLHARLRELL 67% 100%  345 OC1212-14 HIWI/BORISGFTTSILQYENSIMLERSDEIVLTVSNSNV 49% 86% 346 OC1212-15 PRAME/SurvivinRHSQTLKAMVQAWPFAKKVRRAIEQLAAMD 48% 42% *Percentage of individuals havingCD8+ T cell specific PEPI3+ within the Model Population (n = 433).**Percentage of individuals having CD4+ T cell specific PEPI4+ withinnormal donors (n = 400).

Example 19. Efficacy by Design Procedure Exemplified for PolyPEPI1018Colorectal Cancer Vaccine

The PolyPEPI1018 Colorectal Cancer (CRC) Vaccine (PolyPEPI1018)composition is a peptide vaccine intended to be used as an add-onimmunotherapy to standard-of-care CRC treatment options in patientsidentified as likely responders using a companion in vitro diagnostictest (CDx). Clinical trials are ongoing in the US and Italy to evaluatePolyPEPI1018 in metastatic colorectal cancer patients. The productcontains 6 peptides (6 of the 30 mer peptides PolyPEPI1015 described inexamples 16 and 17) mixed with the adjuvant Montanide. The 6 peptideswere selected to induce T cell responses against 12 epitopes from 7cancer testis antigens (CTAs) that are most frequently expressed in CRC.The 6 peptides were optimized to induce long lasting CRC specific T cellresponses. Likely responder patients with T cell responses againstmultiple CTAs expressed in the tumor can be selected with a companiondiagnostic (CDx). This example sets out the precision process used todesign PolyPEPI1018. This process can be applied to design vaccinesagainst other cancers and diseases.

A. Selection of Multiple Antigen Targets

The selection of tumor antigens is essential for the safety and efficacyof cancer vaccines. The feature of a good antigen is to have restrictedexpression in normal tissues so that autoimmunity is prevented. Severalcategories of antigen meet this requirement, including uniquely mutatedantigens (e.g. p53), viral antigens (e.g. human papillomavirus antigensin cervical cancer), and differentiation antigens (e.g. CD20 in B-celllymphoma).

The inventors selected multiple cancer testis antigens (CTAs) as targetantigens since they are expressed in various types of tumor cells andtestis cells, but not expressed in any other normal somatic tissues orcells. CTAs are desirable targets for vaccines for at least thefollowing reasons:

-   -   tumors of higher histological grade and later clinical stage        often show higher frequency of CTA expression    -   only a subpopulation of tumor cells express a certain CTA    -   different cancer types are significantly different in their        frequency of CTA expression    -   tumors that are positive for a CTA often show simultaneous        expression of more than one CTA    -   None of the CTAs appear to be cell surface antigens, therefore        these are unique targets for cancer vaccines (they are not        suitable targets for antibody based immunotherapies)

To identify the target CTAs for PolyPEPI1018, the inventors built a CTAexpression knowledgebase. This knowledgebase contains CTAs that areexpressed in CRC ranked in order by expression rate. Correlation studiesconducted by the inventors (see Example 11) suggest that vaccines whichinduce CTL responses against multiple antigens that are expressed intumor cells can benefit patients. Therefore, seven CTAs with highexpression rates in CRC were selected for inclusion in PolyPEPI1018development. Details are set out in Table 25.

TABLE 25 Target CTAs in PolyPEPI1018 CRC vaccine CTA Expression NameRate Characterization TSP50 89.47% Testis-Specific Protease-Like Protein50 is an oncogene which induces cell proliferation, cell invasion, andtumor growth. It is frequently expressed in gastric-, breast-, cervical-and colorectal cancer samples; and rarely expressed in normal humantissues, except in spermatocytes of testes. EpCAM 88.35% Epithelial CellAdhesion Molecule is a tumor associated antigen, which is expressed incolon cancers and over-expressed in various human carcinomas. The highexpression of EpCAM in cancer-initiating stem cells makes it a valuabletarget for cancer vaccines. EpCAM is also expressed in at low ornegligible levels in normal epithelial cells, with the exception ofsquamous epithelium, hepatocytes and keratinocytes. Survivin 87.28%Survivin (Baculoviral IAP repeat-containing protein 5) is a multi-tasking protein that promotes cell proliferation and inhibits apoptosis.Though it is strongly expressed in fetal tissues and necessary fornormal development, it is not expressed in most adult tissues. Survivinis expressed in various cancers including carcinomas. Normal tissuesthat express low level survivin include thymus, CD34⁺ bone-marrow-derived stem cells, and basal colonic epithelium. Dramatic over-expression of survivin compared with normal tissues iis observed intumors in the lung, breast, colon, stomach, esophagus, pancreas,bladder, uterus, ovaries, large-cell non-Hodgkin's lymphoma, leukemias,neuroblastoma, melanoma and non-melanoma skin cancers. CAGE1 74.47%Cancer-associated gene 1 protein is a typical CTA, which might play arole in cell proliferation and tumorigenesis. CAGE1 is highly expressedin colorectal cancer tissues and weakly expressed in adjacent normalcolorectal mucosa. In addition, CAGE1 is expressed in melanoma,hepatoma, and breast tumors. No CAGE1 protein expression is detected inhealthy human tissues, other than testes. SPAG9 74.36% Sperm-associatedantigen 9 is involved in c-Jun N-terminal kinase- signaling andfunctions as a scaffold protein, thus playing an important role in cellsurvival, proliferation, apoptosis and tumor development. SPAG9expression was detected in epithelial ovarian cancer (90%), breastcancer (88%), cervical cancer (82%), renal cell cancer (88%) andcolorectal cancer (74%) patients. None of the adjacent noncanceroustissues showed antigen expression. SPAG9 expression is restricted totestis. FBXO39 38.60% FBXO39 (BCP-20) is a testis specific protein andis an important part of the E3 ubiquitin ligase complex. It participatesin ubiquitination and has a role in regulating the cell cycle, immuneresponses, signaling, and proteasomal degradation of proteins. FBXO39 isexpressed in colon and breast cancers. FBXO39 expression has also beendetected in ovary, placenta, and lung. FBXO39 expression is 100-foldhigher in testis and 1,000-fold higher in colorectal cancers comparedwith normal tissue. MAGEA8 43.75% Melanoma-associated antigen 8 functionis not known, though it may play a role in embryonal development andtumor transformation or aspects of tumor progression. MAGE-A8 gene isexpressed in CRC and hepatocellular carcinoma. MAGE-A8 expression innormal tissues is restricted to the testis and the placenta.B. Precise Targeting is Achieved by PEPI3+ Biomarker Based VaccineDesign

As described above the PEPI3+ biomarker predicts a subject's vaccineinduced T cell responses. The inventors developed and validated a testto accurately identify the PEPIs from antigen sequences and HLAgenotypes (Examples 1, 2, 3). The PEPI Test algorithm was used toidentify the dominant PEPIs (besEPIs) from the 7 target CTAs to beincluded in PolyPEPI1018 CRC vaccine.

The dominant PEPIs identified with the process described here can induceCTL responses in the highest proportion of subjects:

-   -   i. Identification of all HLA class I binding PEPIs from the 7        CTA targets in each of the 433 subjects in the Model Population    -   ii. Identification of the dominant PEPIs (BestEPIs) that are        PEPIs present in the largest subpopulation.

The 12 dominant PEPIs that are derived from the 7 CTAs in PolyPEPI1018are presented in the Table 26. The PEPI % in Model Population indicatesthe proportion of 433 subjects with the indicated PEPI, i.e. theproportion of subjects where the indicated PEPI can induce CTLresponses. There is very high variability (18%-78%) in the dominantPEPIs to induce CTL responses despite the optimization steps used in theidentification process.

TABLE 26 CRC specific HLA class I binding dominant PEPIsin PolyPEPI1018 Dominant PEPI3+ for each of the7 CTAs in PolyPEPI1018 in CRC patients PEPI3+ % Peptides in CRC Dominantin Model PolyPEPI1018 Antigens PEPI3+ Population CRC-P1 TSP50 TTMETQFPV36% YRAQRFWSW 20% CRC-P2 EpCAM RTYWIIIEL 51% Survivin RAIEQLAAM 26%CRC-P3 EpCAM YVDEKAPEF 28% MAGE-A8 KVAELVRFL 18% CRC-P6 CAGE1 KMHSLLALM42% Survivin STFKNWPFL 15% CRC-P7 CAGE1 KSMTMMPAL 37% SPAG9 VMSERVSGL28% CRC-P8 FBXO39 FMNPYNAVL 78% FFFERIMKY 46%

The inventors optimized each dominant PEPI to bind to most HLA class IIalleles of most subjects. This should enhance efficacy, because it willinduce CD4⁺ T helper cells that can augment CD8⁺ CTL responses andcontribute to long lasting T cell responses. The example presented inFIG. 4 demonstrates that PEPIs that bind to ≥3 HLA class II alleles mostlikely activate T helper cells.

The 15-mer peptides selected with the process described here containboth HLA class I and class II binding dominant PEPIs. Therefore, thesepeptides can induce both CTL and T helper responses in the highestproportion of subjects.

Process:

-   -   1. Identification the HLA class II genotype of 400 normal        donors*    -   2. Extension of each 9-mer dominant PEPI (Table 20) on both        sides with amino acids that match the source antigen    -   3. Prediction of HLA class II PEPIs of 400 normal donors using        an IEDB algorithm    -   4. Selection the 15-mer peptide with the highest proportion of        subject have HLA Class II binding PEPIs    -   5. Ensure the presence of one dominant HLA class II PEPI in each        vaccine peptide when joining two 15-mer peptides

The 12 optimized 15-mer peptides derived from the 7 CTAs in PolyPEPI1018are presented in the Table 27. These peptides have different HLA classII binding characteristics. There is a high variability (0%-100%) inPEPI generation capacity (HLA binding) among these peptides despite suchan optimized personalized vaccine design.

TABLE 27 Antigen specific HLA class II binding PEPIs in PolyPEPI1018.Average % subjects % subjects % subjects % subjects HLA class with ≥1with ≥2 with ≥3 with ≥4 II binding HLA class HLA class HLA class HLAclass Nr. CRC antigens alleles II binding II binding II binding IIbinding CRC-P1 TSP50 (83-97) 0  0%  0%  0%  0% TSP50 (190-204) 4 100%99% 88% 53% CRC-P2 EPCAM(139-153) 5 100% 100%  100%  98%SURVIVIN(127-141) 2  84% 58% 26% 11% CRC-P3 EPCAM(251-265) 0  0%  0%  0% 0% MAGE-A8(113-127) 4 100% 100%  95% 72% CRC-P6 CAGE1(613-627) 5 100%100%  99% 95% SURVIVIN(15-29) 3 100% 97% 83% 45% CRC-P7 CAGE1(759-773) 3100% 98% 87% 56% SPAG9(16-30) 1  66% 35%  9%  2% CRC-P8 FBXO39(95-109) 3100% 94% 43% 13% FBXO39(284-298) 5 100% 100%  100%  98%

The 30-mer vaccine peptides have the following advantages compared toshorter peptides:

-   -   (i) Multiple precisely selected tumor specific immunogens: each        30 mer contains two precisely selected cancer specific        immunogenic peptides that are capable to induce CTL and T helper        responses in the majority of the relevant population (similar to        the model population).    -   (ii) Ensure natural antigen presentation. 30-mer long        polypeptides can be viewed as pro-drugs: They are not        biologically active by themselves, but are processed to smaller        peptides (9 to 15 amino acid long) to be loaded into the HLA        molecules of professional antigen presenting cells. The antigen        presentation resulting from long peptide vaccination reflects        physiological pathways for presentation in both HLA class I and        class II molecules. In addition, long peptide processing in the        cells is much more efficient than that of large intact proteins.    -   (iii) Exclude induction of tolerizing T cell responses. 9-mer        peptides do not require processing by professional        antigen-presenting cells and therefore bind exogenously to the        HLA class I molecules. Thus, injected short peptides will bind        in large numbers to HLA class I molecules of all nucleated cells        that have surface HLA class I. In contrast, >20-mers long        peptides are processed by antigen presenting cells before        binding to HLA class I. Therefore, vaccination with long        peptides is less likely to lead to tolerance and will promote        the desired antitumor activity.    -   (iv) Induce long lasting T cell responses because it can        stimulate T helper responses by binding to multiple HLA class II        molecules    -   (v) Utility. GMP manufacturing, formulation, quality control and        administration of a smaller number of peptides (each with all of        the above characteristics) is more feasible than a larger number        of peptides supplying different characteristics.

Each 30-mer peptide in PolyPEPI1018 consists of 2 HLA class I bindingdominant PEPIs and at least one strong HLA class II binding PEPI. Strongbinding PEPIs bind to 4 HLA class II alleles in >50% of individuals.Therefore, the vaccine peptides are tailored to both HLA class I andclass II alleles of individual subjects in a general population (whichis a relevant population for CRC vaccine design).

As demonstrated above the high HLA genotype variability in subjectsresults in high variability of T cell responses induced by PolyPEPI1018.This justifies the co-development of a CDx that determines likelyresponders. The PEPI3+ and >2PEPI3+ biomarkers could predict the immuneresponse and clinical responses, respectively, of subjects vaccinatedwith PolyPEPI1018 as detailed in Examples 11 and 12. These biomarkerswill be used to co-develop a CDx which predicts likely responders toPolyPEPI1018 CRC vaccine.

Example 20—Analysis of the Composition and Immunogenicity ofPolyPEPI1018 CRC Vaccine

Selected peptides for the PolyPEPI1018 composition are as shown in Table28.

TABLE 28Selected Colorectal Cancer Vaccine peptides for PolyPEPI1018 compositionHLAI* HLAII** SEQID TREOSID Source Antigen Peptide (30mer) (CD8) (CD4)130 CCV1000-5-1 TSP50 PSTTMETQFPVSEGKSRYRAQRFWSWVGQA 53% 88% 121CCV1000-2-2 EpCAM/Survivin VRTYWIIIELKHKARTAKKVRRAIEQLAAM 57% 100%  131CCV1000-5-3 EpCAM/Mage-A8 YVDEKAPEFSMQGLKDEKVAELVRFLLRKY 43% 95% 124CCV1000-2-6 Cage/Survivin LASKMHSLLALMVGLKDHRISTFKNWPFLE 58% 99% 134CCV1000-5-7 Cage/Spag9 PKSMTMMPALFKENRSGAVMSERVSGLAGS 57% 87% 126CCV1000-2-8 FBXO39 KFMNPYNAVLTKKFQKVNFFFERIMKYERL 90% 100% PolyPEPI1018 (6 peptide together) 98% 100%  *Percentage of individualshaving HLA class I binding PEPI3+ within the Model Population (n = 433).**Percentage of individuals having HLA class II binding PEPI3+ withinthe Model Population (n = 433).

The peptides of PolyPEPI1018 are formulated in two mixtures, MIX1containing the peptides of SEQ ID: 130, 131 and MIX2 containing thepeptides of SEQ ID: 121, 124, 134, 126. MIX 1 and MIX 2 may beadministered sequentially.

Characterization of Immunogenicity

The inventors used the PEPI3+ Test to characterized the immunogenicityof PolyPEPI1018 in a cohort of 37 CRC patients with complete HLAgenotype data. T cell responses were predicted in each patient againstthe same 9 mer peptides that will be used in clinical trials. Thesepeptides represent the 12 dominant PEPI3+ within the PolyPEPI1018peptides. The 9 mers are shown in Table 26.

The specificity and sensitivity of PEPI3+ prediction depends on theactual number of HLAs predicted to bind a particular epitope.Specifically, the inventors have determined that the probability thatone HLA-restricted epitope induces a T cell response in a subject istypically 4%, which explains the poor sensitivity of the state-of-artprediction methods based on HLA restricted epitope prediction. Applyingthe PEPI3+ methodology, the inventors determined the probability that Tcell response to each of the dominant PEPI3+-specific would be inducedby PolyPEPI1018 in the 37 CRC patients. The results from this analysisare summarized in the Table 29.

TABLE 29 Probability of Dominant PEPI in the 6 Peptides of PolyPEPI1018in 37 CRC Patients CRC-P1 CRC-P2 CRC-P3 CRC-P6 CRC-P7 CRC-P8 TSP50 EpCAMSurvivin EpCAM MAGEA8 CAGE1 CAGE1 FBXO39 FBXO39 Expected CRC TSP50 (190-(139- (127- (251- (113- (613- Survivin (759- SPAG9 (95- (284- Number ofPatient (83-97) 204) 153) 141) 265) 127) 627) (15-29) 773) (16-30) 109)298) PEPIs CRC-01 22%  4% 22%   4% 22% 22%  100%  1% 98%  84%  100% 22%5.01 CRC-02 22%  1% 22%  22% 22% 22%  100%  1% 98%  22%  100% 98% 5.29CRC-03 84% 22% 84%  22% 22% 22%  84% 22%  22%  22%  100% 22% 5.29 CRC-0422% 84% 22%   4% 22% 4% 98% 4% 4% 22%  100% 84% 4.70 CRC-05 22% 22% 4% 4% 22% 4% 98% 1% 4% 4% 100% 84% 3.68 CRC-06 84% 22% 4% 84% 98% 4% 22%4% 4% 4% 100% 98% 5.27 CRC-07 22% 22% 22%  22% 22% 4% 98% 1% 22%  22% 100% 84% 4.41 CRC-08 22% 22% 22%  98% 84% 22%  84% 22%  22%  22%  100%84% 6.04 CRC-09 22% 84% 84%  84% 84% 22%  100%  4% 22%  22%   98% 84%7.10 CRC-10  4% 98% 22%  22%  4% 4%  4% 22%  22%  22%   98% 84% 4.06CRC-11 22% 22% 4%  4% 22% 4% 84% 1% 4% 4%  98% 84% 3.53 CRC-12 84% 22%4% 22%  4% 4% 84% 4% 84%  4% 100% 22% 4.38 CRC-13 84% 22% 4% 22% 84% 4%84% 1% 1% 4% 100% 98% 5.07 CRC-14 22% 84% 4%  4% 22% 4% 84% 1% 4% 4%100% 84% 4.16 CRC-15 84% 22% 22%  22% 22% 4% 84% 4% 22%  4% 100% 84%4.74 CRC-16  4% 84% 4%  4% 22% 4% 84% 1% 4% 22%  100% 84% 4.16 CRC-1784% 84% 4% 84% 84% 4%  4% 4% 4% 4% 100% 22% 4.82 CRC-18 84% 22% 22%  84%84% 4% 22% 22%  4% 4% 100% 84% 5.36 CRC-19 22% 22% 22%  22% 22% 4% 98%4% 22%  22%  100% 84% 4.45 CRC-20 84% 22% 4% 22% 84% 4% 84% 1% 4% 4%100% 98% 5.10 CRC-21 22% 22% 22%  22% 84% 22%  98% 4% 4% 22%  100% 84%5.06 CRC-22 22% 98% 84%   4% 22% 22%  84% 22%  84%  22%   98% 22% 5.84CRC-23 84% 84% 84%  84% 84% 22%  84% 84%  84%  4% 100% 84% 8.82 CRC-2422% 22% 4%  4% 22% 4% 84% 1% 4% 4% 100% 84% 3.55 CRC-25 22% 84% 22%   4%22% 4% 84% 4% 22%  4% 100% 84% 4.56 CRC-26 84% 22% 4% 22% 84% 4% 84% 1%4% 4% 100% 84% 4.97 CRC-27 22% 22% 4%  4% 22% 4% 98% 1% 4% 4% 100% 84%3.68 CRC-28 84% 22% 4% 22% 84% 4% 84% 1% 4% 4% 100% 98% 5.10 CRC-29 84%84% 4% 22% 22% 4% 84% 1% 22%  22%  100% 84% 5.33 CRC-30 84% 22% 4% 22%84% 4% 84% 1% 4% 4% 100% 98% 5.10 CRC-31 22% 84% 22%   4%  4% 4% 22% 1%4% 4%  98% 84% 3.53 CRC-32 84% 84% 4% 84% 22% 4%  4% 4% 4% 4%  98% 84%4.80 CRC-33 84% 22% 4% 22% 84% 4% 84% 1% 4% 4% 100% 98% 5.10 CRC-34 22%22% 22%  22% 22% 4% 84% 1% 22%  4% 100% 84% 4.09 CRC-35 22%  4% 4%  1%22% 4%  4% 1% 4% 4%  84% 84% 2.37 CRC-36 22%  4% 4%  1% 22% 4%  4% 1% 4%4%  84% 84% 2.37 CRC-37 22%  4% 4%  1% 22% 4%  4% 1% 4% 4%  84% 84% 2.37Abbreviations: CRC = colorectal cancer; PEPI = personal epitope Note:Percentages represent the likelihood of CD8+ T cell Responses Induced byPolyPEPI1018.

Overall, these results show that the most immunogenic peptide inPolyPEPI1018 is CRC-P8, which it is predicted to bind to >3 HLAs in mostpatients. The least immunogenic peptide, CRC-P3, binds to >1 HLA in manypatients and has a 22% chance of inducing T cell responses. Sincebioassays used to detect T cell responses are less accurate than PEPI3+,this calculation may be the most accurate characterization of the T cellresponses in CRC patients. Though MAGE-A8 and SPAG9 were immunogenic inthe Model Population used for vaccine design, MAGE-A8-specific PEPI3+were absent in the 37 CRC patients, and only one patient (3%) had SPAG9specific PEPI3+.

Further characterization of the predicted PolyPEPI1018 response rate inthe model population described in Example 8 and in 295 CRC patients withknown HLA class I genotypes are shown in Tables 30 and 31.

TABLE 30 PolyPEPI1018 Response Rates in the Model Population (433 Normaldonors) PolyPEPI1018 Response Rates >=1 >=2 >=3 >=4 >=5 >=6 >=7 >=8 >=9Multi PEPI 98% 94% 83% 70% 52% 38% 27% 18% 11% Multi Peptide 98% 91% 73%52% 30% 12% N/D N/D N/D Multi Antigen 98% 92% 72% 49% 31% 14%  6% N/DN/D

TABLE 31 PolyPEPI1018 Response Rates for 295 CRC patients PolyPEPI1018Response Rates >=1 >=2 >=3 >=4 >=5 >=6 >=7 >=8 >=9 Multi PEPI 99% 96%92% 85% 69% 53% 40% 32% 25% Multi Peptide 99% 93% 86% 71% 49% 29% N/DN/D N/D Multi Antigen 99% 93% 86% 72% 49% 32% 13% N/D N/DCharacterization of Toxicity—immunoBLAST

A method was developed that can be performed on any antigen to determineits potential to induce toxic immune reaction, like autoimmunity. Themethod is referred to herein as immunoBLAST. PolyPEPI1018 contains six30-mer polypeptides. Each polypeptide consists of two 15-mer peptidefragments derived from antigens expressed in CRC. Neoepitopes might begenerated in the joint region of the two 15-mer peptides and couldinduce undesired T cell responses against healthy cells (autoimmunity).This was assesses using the immunoBLAST methodology.

A 16-mer peptide for each of the 30-mer components of PolyPEP1018 wasdesigned. Each 16-mer contains 8 amino acids from the end of the first15 residues of the 30-mer and 8 amino acids from the beginning of thesecond 15 residues of the 30-mer—thus precisely spanning the jointregion of the two 15-mers. These 16-mers are then analysed to identifycross-reactive regions of local similarity with human sequences usingBLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), which compares proteinsequences to sequence databases and calculates the statisticalsignificance of matches. 8-mers within the 16-mers were selected as theexamination length since that length represents the minimum lengthneeded for a peptide to form an epitope, and is the distance between theanchor points during HLA binding.

As shown in FIG. 19 , the positions of amino acids in a polypeptide arenumbered. The start positions of potential 9-mer peptides that can bindto HLAs and form neoepitopes are the 8 amino acids in positions 8-15.The start positions of tumor antigen derived peptides harbored by the15-mers that can form the pharmaceutically active epitopes are 7+7=14amino acids at position 1-7 and 16-22. The ratio of possible neoepitopegenerating peptides is 36.4% ( 8/22).

The PEPI3+ Test was to identify neoepitopes and neoPEPI among the 9-merepitopes in the joint region. The risk of PolyPEPI1018 inducing unwantedT cell responses was assessed in the 433 subjects in the ModelPopulation by determining the proportion of subjects with PEPI3+ amongthe 9-mers in the joint region. The result of neoepitope/neoPEPIanalysis is summarized in Table 32. In the 433 subjects of the ModelPopulation, the average predicted epitope number that could be generatedby intracellular processing was 40.12. Neoepitopes were frequentlygenerated; 11.61 out of 40.12 (28.9%) epitopes are neoepitopes. Most ofthe peptides were able to be identified as a neoepitope, but the numberof subjects that present neoepitopes varied.

Epitopes harbored by PolyPEPI1018 create an average of 5.21 PEPI3+.These PEPIs can activate T cells in a subject. The amount of potentialneoPEPIs was much lower than neoepitopes (3.7%). There is a marginalpossibility that these neoPEPIs compete on T cell activation with PEPIsin some subjects. Importantly, the activated neoPEPI specific T cellshad no targets on healthy tissue.

TABLE 32 Identification of Potential Neoepitopes of PolyPEPI1018Epitope & PEPI3+ binding in 433 Subjects of the Model PopulationEpitope Binding (1 × HLA) PEPI3+ binding (3 × HLA) PolyPEPI1018Potential NeoEPI NeoPEPI Peptide ID: Neoepitope Sub# Sub % NeoEPI countSub# Sub % NeoPEPI count CRC-P1 QFPVSEGKS 0 0.0% 7 0 0.0% 3 FPVSEGKSR160 37.0% X 1 0.2% X PVSEGKSRY 150 34.6% X 0 0.0% VSEGKSRYR 194 44.8% X1 0.2% X SEGKSRYRA 113 26.1% X 0 0.0% EGKSRYRAQ 77 17.8% X 0 0.0%GKSRYRAQR 37 8.5% X 0 0.0% KSRYRAQRF 337 77.8% X 33 7.6% X CRC-P2IELKHKART 32 7.4% X 7 0 0.0% 1 ELKHKARTA 63 14.5% X 0 0.0% LKHKARTAK 5913.6% X 0 0.0% KHKARTAKK 166 38.3% X 1 0.2% X HKARTAKKV 0 0.0% 0 0.0%KARTAKKVR 70 16.2% X 0 0.0% ARTAKKVRR 134 30.9% X 0 0.0% RTAKKVRRA 419.5% X 0 0.0% CRC-P3 EFSMQGLKD 0 0.0% 5 0 0.0% 1 FSMQGLKDE 188 43.4% X 00.0% SMQGLKDEK 138 31.9% X 0 0.0% MQGLKDEKV 16 3.7% X 0 0.0% QGLKDEKVA 00.0% 0 0.0% GLKDEKVAE 0 0.0% 0 0.0% LKDEKVAEL 186 43.0% X 3 0.7% XKDEKVAELV 51 11.8% X 0 0.0% CRC-P6 LLALMVGLK 252 58.2% X 7 0 0.0% 1LALMVGLKD 86 19.9% X 0 0.0% ALMVGLKDH 65 15.0% X 0 0.0% LMVGLKDHR 9722.4% X 0 0.0% MVGLKDHRI 67 15.5% X 0 0.0% VGLKDHRIS 0 0.0% 0 0.0%GLKDHRIST 4 0.9% X 0 0.0% LKDHRISTF 195 45.0% X 5 1.2% X CRC-P7PALFKENRS 0 0.0% 5 0 0.0% 1 ALFKENRSG 0 0.0% 0 0.0% LFKENRSGA 41 9.5% X0 0.0% FKENRSGAV 114 26.3% X 0 0.0% KENRSGAVM 261 60.3% X 0 0.0%ENRSGAVMS 0 0.0% 0 0.0% NRSGAVMSE 227 52.4% X 0 0.0% RSGAVMSER 197 45.5%X 2 0.5% X CRC-P8 AVLTKKFQK 181 41.8% X 7 0 0.0% 3 VLTKKFQKV 208 48.0% X2 0.5% X LTKKFQKVN 0 0.0% 0 0.0% TKKFQKVNF 25 5.8% X 0 0.0% KKFQKVNFF250 57.7% X 12 2.8% X KFQKVNFFF 273 63.0% X 23 5.3% X FQKVNFFFE 16337.6% X 0 0.0% QKVNFFFER 110 25.4% X 0 0.0% Abbreviations: CRC= colorectal cancer; HLA = human leukocytic antigen; PEPI = personalepitope

Each of the 30-mer peptides in PolyPEPI1018 were released for clinicaldevelopment since none of the 8-mers in the joint regions matched anyhuman protein, except the target CTAs.

Characterisation of Activity/Efficacy

The inventors have developed pharmacodynamic biomarkers to predict theactivity/effect of vaccines in individual human subjects as well as inpopulations of human subjects. These biomarkers expedite more effectivevaccine development and also decrease the development cost. Theinventors have the following tools:

Antigen expression knowledgebase: The inventors have collected data fromexperiments published in peer reviewed scientific journals regarding thetumor antigens expressed by tumor cells and organized by tumor type tocreate a database of CTA expression levels—CTA database (CTADB). As ofApril 2017, the CTADB contained data from 145 CTAs from 41,132 tumorspecimens, and was organized by the CTA expression frequencies indifferent types of cancer.In silico trial populations: The inventors have also collected data onthe HLA genotypes of several different model populations. Eachindividual in the populations has complete 4-digit HLA genotype andethnicity data. The populations are summarized in Table 33.

TABLE 33 In silico trial populations Number of Population SubjectsInclusion criteria Model Population 433 Complete HLA class I genotypeDiverse ethnicity CRC patients 37 Complete HLA class I genotype CRCdiagnosis, unknown ethnicity “Big” Population 7,189 Complete HLA class Igenotype Diverse ethnicity Chinese Population 234 Complete HLA class Igenotype Chinese ethnicity Irish Population 999 Complete HLA class Igenotype Irish ethnicity Abbreviations: CRC = colorectal cancer; HLA =human leukocyte antigen

Using these tools (or potentially equivalent databases or modelpopulations), the following markers can be assessed:

-   -   AG95—potency of a vaccine: The number of antigens in a cancer        vaccine that a specific tumor type expresses with 95%        probability. AG95 is an indicator of the vaccine's potency, and        is independent of the immunogenicity of the vaccine antigens.        AG95 is calculated from the tumor antigen expression rate data,        which is collected in the CTADB. Technically, AG95 is determined        from the binomial distribution of CTAs, and takes into account        all possible variations and expression rates. In this study,        AG95 was calculated by cumulating the probabilities of a certain        number of expressed antigens, by the widest range of antigens        where the sum of probabilities was less than or equal to 95%.        The correct value is between 0 (no expression expected with 95%        probability) and maximum number of antigens (all antigens        expressed with 95% probability).    -   PEPI3+ count—immunogenicity of a vaccine in a subject:        Vaccine-derived PEPI3+ are personal epitopes that induce T cell        responses in a subject. PEPI3+ can be determined using the        PEPI3+ Test in subjects who's complete 4-digit HLA genotype is        known.    -   AP count—antigenicity of a vaccine in a subject: Number of        vaccine antigens with PEPI3+. Vaccines like PolyPEPI1018 contain        sequences from antigens expressed in tumor cells. AP count is        the number of antigens in the vaccine that contain PEPI3+, and        the AP count represents the number of antigens in the vaccine        that can induce T cell responses in a subject. AP count        characterizes the vaccine-antigen specific T cell responses of        the subject since it depends only on the HLA genotype of the        subject and is independent of the subject's disease, age, and        medication. The correct value is between 0 (no PEPI presented by        the antigen) and maximum number of antigens (all antigens        present PEPIs).    -   AP50—antigenicity of a vaccine in a population: The mean number        of vaccine antigens with a PEPI in a population. The AP50 is        suitable for the characterization of vaccine-antigen specific T        cell responses in a given population since it depends on the HLA        genotype of subjects in a population. Technically, the AP count        was calculated in the Model Population and the binomial        distribution of the result was used to calculate the AP50.    -   AGP count—effectiveness of a vaccine in a subject: Number of        vaccine antigens expressed in the tumor with PEPI. The AGP count        indicates the number of tumor antigens that vaccine recognizes        and induces a T cell response against (hit the target). The AGP        count depends on the vaccine-antigen expression rate in the        subject's tumor and the HLA genotype of the subject. The correct        value is between 0 (no PEPI presented by expressed antigen) and        maximum number of antigens (all antigens are expressed and        present a PEPI).    -   AGP50—effectiveness of a cancer vaccine in a population: The        mean number of vaccine antigens expressed in the indicated tumor        with PEPI (i.e., AGP) in a population. The AGP50 indicates the        mean number of tumor antigens that the T cell responses induced        by the vaccine can recognize. AGP50 is dependent on the        expression rate of the antigens in the indicated tumor type and        the immunogenicity of the antigens in the target population.        AGP50 can estimate a vaccine's effectiveness in different        populations and can be used to compare different vaccines in the        same population. The computation of AGP50 is similar to that        used for AG50, except the expression is weighted by the        occurrence of the PEPI3+ in the subject on the expressed vaccine        antigens. In a theoretical population, where each subject has a        PEPI from each vaccine antigen, the AGP50 will be equal to AG50.        In another theoretical population, where no subject has a PEPI        from any vaccine antigen, the AGP50 will be 0. In general, the        following statement is valid: 0≤AGP50≤AG50.    -   mAGP—a candidate biomarker for the selection of likely        responders: Likelihood that a cancer vaccine induces T cell        responses against multiple antigens expressed in the indicated        tumor. mAGP is calculated from the expression rates of        vaccine-antigens in CRC and the presence of vaccine derived        PEPIs in the subject. Technically, based on the AGP        distribution, the mAGP is the sum of probabilities of the        multiple AGP (≥2 AGPs).        Application of these Markers to Assess Antigenicity and        Effectiveness PolyPEPI1018 in Individual Patients with CRC

Table 34 shows the antigenicity and effectiveness of PolyPEPI1018 in 37CRC patients using AP and AGP50, respectively. As expected from the highvariability of PolyPEPI1018 specific T cell responses (see Table 29),the AP and AGP50 have high variability. The most immunogenic antigen inPolyPEPI1018 was FOX039; each patient had a PEPI3+. However, FOX039 isexpressed only 39% of CRC tumors, suggesting that 61% of patients willhave FOX039 specific T cell responses that do not recognize the tumor.The least immunogenic antigen was MAGE-A8; none of the 37 CRC patientshad a PEPI3+ despite the antigen being expressed in 44% of CRC tumors.These results illustrate that both expression and immunogenicity ofantigens can be taken into account when determining a cancer vaccine'seffectiveness.

AGP50 indicates the mean number of expressed antigens in CRC tumor withPEPIs. Patients with higher AGP50 values are more likely to respond toPolyPEPI1018 since higher AGP50 values indicate that the vaccine caninduce T cell responses against more antigens expressed in CRC cells.

The last column in the table 32 shows the probability of mAGP (multipleAGP; i.e., at least 2 AGPs) in each of the 37 CRC patients. The averagemAGP in patients with CRC is 66%, suggesting that there is a 66%likelihood that a CRC patient will induce T cell responses againstmultiple antigens expressed in the tumor.

TABLE 34 Antigenicity (AP count), Effectiveness (AGP50 count), and mAGPof PolyPEPI1018 in 37 CRC Patients Antigens (CTAs) in PolyPEPI1018Number of TSP50 EpCAM Survivin CAGE1 SPAG9 FBXO39 MAGE-A8 Number ofAGP50 CRC Expression rate AP (AP (AGP50 Patients 89% 88% 87% 74% 74% 39%44% count) count) mAGP CRC-01 0 0 0 1 1 1 0 3 1.87 90% CRC-02 0 0 0 1 01 0 2 1.13 85% CRC-03 1 1 0 1 0 1 0 4 2.91 97% CRC-04 1 0 0 1 0 1 0 32.03 91% CRC-05 0 0 0 1 0 1 0 2 1.13 78% CRC-06 1 1 1 1 0 1 0 5 3.78 99%CRC-07 0 0 0 1 0 1 0 2 1.13 84% CRC-08 0 1 1 1 0 1 0 4 2.89 98% CRC-09 11 1 1 0 1 0 5 3.78 99% CRC-10 1 0 0 0 0 1 0 2 1.28 86% CRC-11 0 0 0 1 01 0 2 1.13 79% CRC-12 1 0 0 1 0 1 0 3 2.03 88% CRC-13 1 1 1 1 0 1 0 53.78 98% CRC-14 1 0 0 1 0 1 0 3 2.03 87% CRC-15 1 0 0 1 0 1 0 3 2.03 90%CRC-16 1 0 0 1 0 1 0 3 2.03 85% CRC-17 1 1 1 0 0 1 0 4 3.04 96% CRC-18 11 1 1 0 1 0 5 3.78 98% CRC-19 0 0 0 1 0 1 0 2 1.13 85% CRC-20 1 1 1 1 01 0 5 3.78 98% CRC-21 0 1 0 1 0 1 0 3 2.01 93% CRC-22 1 1 0 1 0 1 0 42.91 97% CRC-23 1 1 1 1 0 1 0 5 3.78 99% CRC-24 0 0 0 1 0 1 0 2 1.13 82%CRC-25 1 0 0 1 0 1 0 3 2.03 89% CRC-26 1 1 0 1 0 1 0 4 2.91 95% CRC-27 00 0 1 0 1 0 2 1.13 78% CRC-28 1 1 1 1 0 1 0 5 3.78 98% CRC-29 1 0 0 1 01 0 3 2.03 92% CRC-30 1 1 1 1 0 1 0 5 3.78 98% CRC-31 1 0 0 0 0 1 0 21.28 80% CRC-32 1 0 1 0 0 1 0 3 2.15 91% CRC-33 1 1 1 1 0 1 0 5 3.78 98%CRC-34 0 0 0 1 0 1 0 2 1.13 82% CRC-35 0 0 0 0 0 1 0 1 0.39 55% CRC-36 00 0 0 0 1 0 1 0.39 55% CRC-37 0 0 0 0 0 1 0 1 0.39 55% Abbreviations:CRC = colorectal cancer; PEPI = personal epitope; CTA = cancer testisantigen; AP = expressed antigens with ≥1 PEPI

These biomarkers have immediate utility in vaccine development and inthe routine clinical practice because they do not require invasivebiopsies. Antigen expression data can be obtained from achieved tumorspecimen and organized in databases. 4-digit HLA genotyping can be donefrom a saliva specimen. It is a validated test performed by certifiedlaboratories worldwide for transplantation and paternity testing. Theseassessments will allow drug developers and physicians to gain deeperinsights into the immunogenicity and activity of tumor response and thepossible emergence of resistance.

Application of these Markers to Asses Antigenicity and EffectivenessPolyPEPI1018 in Populations

Antigenicity of PolyPEPI1018 CRC Vaccine in a General Population

The antigenicity of PolyPEPI1018 in a subject is determined by the APcount, which indicates the number of vaccine antigens that induce T cellresponses in a subject. The AP count of PolyPEPI1018 was determined ineach of the 433 subjects in the Model Population using the PEPI Test,and the AP50 count was then calculated for the Model Population.

As shown in FIG. 20 the AP50 of PolyPEPI1018 in the Model Population is3.62. Therefore, the mean number of immunogenic antigens (i.e., antigenswith ≥1 PEPI) in PolyPEPI1018 in a general population is 3.62.

Effectiveness of PolyPEPI1018 CRC Vaccine in a General Population

Vaccine induced T cells can recognize and kill tumor cells if a PEPI inthe vaccine is presented by the tumor cell. The number of AGPs(expressed antigens with PEPI) is an indicator of vaccine effectivenessin an individual, and is dependent on both the potency and antigenicityof PolyPEPI1018. As shown in FIG. 21 , the mean number of immunogenicCTAs (i.e., AP [expressed antigens with ≥1 PEPI]) in PolyPEPI1018 is2.54 in the Model Population. The likelihood that PolyPEPI1018 induces Tcell responses against multiple antigens in a subject (i.e., mAGP) inthe Model Population is 77%.

Comparison of the PolyPEPI1018 CRC Vaccine Activities in DifferentPopulations

Tables 35 to 37 show comparison of the immunogenicity, antigenicity, andeffectiveness of PolyPEPI1018 in different populations.

TABLE 35 Comparison of Immunogenicity, Antigenicity, and Effectivenessof PolyPEPI1018 in Different Sub-populations Number of Number of PEPI3+Number of AP Number of AGP50 Populations subject Average SD Average SDAverage SD CRC 37 5.16 1.98 3.19 1.31 2.21 1.13 Model 433 5.02 2.62 3.621.67 2.54 1.25 Big 7,189 5.20 2.82 3.75 1.74 2.66 1.30 Chinese 324 5.973.16 4.28 1.78 3.11 1.30 Irish 999 3.72 1.92 2.86 1.46 1.94 1.10Abbreviations: CRC = colorectal cancer; PEPI = personal epitope; SD =standard deviation; AP = expressed antigens with ≥1 PEPI

The average number of PEPI3+ and AP results demonstrate thatPolyPEPI1018 is highly immunogenic and antigenic in all populations;PolyPEPI1018 can induce an average of 3.7-6.0 CRC specific T cell clonesagainst 2.9-3.7 CRC antigens. PolyPEPI1018 immunogenicity was similar inpatients with CRC and the average population (p>0.05), this similaritymay have been due to the small sample size of the CRC population.Additional analyses suggest that PolyPEPI1018 is significantly moreimmunogenic in a Chinese population compared to an Irish or a generalpopulation (p<0.0001). The differences in immunogenicity are alsoreflected in the effectiveness of the vaccine as characterized by AGP50;PolyPEPI1018 is most effective in a Chinese population and lesseffective in an Irish population. Since a CDx will be used to selectlikely responders to PolyPEPI1018, ethnic differences will only bereflected in the higher percentage of Chinese individuals that might beeligible for treatment compared with Irish individuals.

TABLE 36 PolyPEPI1018 CRC Vaccine, Predicted Immune Response RatesAgainst Multiple CRC Antigens No. PolyPEPI1018 MultiAG CTL ResponsesPopulation subjects ≥3 ≥4 ≥5 ≥6 7 CRC Vietnamese 211 91% 81% 56% 38% 17%Patients US 44 57% 34% 20%  5%  0% Caucasian 83 75% 51% 30% 17%  4%Normal US 400 61% 39% 25% 12%  3% Donors Europe 1,386 55% 30% 18%  7% 1% Chinese 324 84% 68% 45% 26% 15% Okinawan (JP) 104 81% 57% 36% 16%13% Japanese 45 77% 55% 34% 16% 13%

TABLE 37 PolyPEPI1018 CRC Vaccine, Predicted Immune Response RatesAgainst Multiple CRC Antigens No. Number of PEPI Number of AP Number ofAGP50 Population subjects Average SD Average SD Average SD CRCVietnamese 211 6.96 3.01 4.81 1.58 3.47 1.16 Patients US 44 4.05 2.053.00 1.46 2.05 1.12 Caucasian 83 4.75 2.39 3.57 1.76 2.50 1.27 Normal US400 4.30 2.50 3.19 1.74 2.17 1.30 Donors Europe 1,386 3.84 2.01 2.941.51 2.00 1.14 Chinese 324 5.97 3.16 4.28 1.78 3.11 1.30 Okinawan (JP)104 5.29 2.58 4.01 1.63 2.91 1.19 Japanese 45 5.31 3.27 3.67 1.77 2.661.29

Example 21—Personalised Immunotherapy Composition for Treatment ofOvarian Cancer

This example describes the treatment of an ovarian cancer patient with apersonalised immunotherapy composition, wherein the composition wasspecifically designed for the patient based on her HLA genotype based onthe disclosure described herein. This Example and Example 22 belowprovide clinical data to support the principals regarding binding ofepitopes by multiple HLA of a subject to induce a cytotoxic T cellresponse on which the present disclosure is based.

The HLA class I and class II genotype of metastatic ovarianadenocarcinoma cancer patient XYZ was determined from a saliva sample.

To make a personalized pharmaceutical composition for patient XYZthirteen peptides were selected, each of which met the following twocriteria: (i) derived from an antigen that is expressed in ovariancancers, as reported in peer reviewed scientific publications; and (ii)comprises a fragment that is a T cell epitope capable of binding to atleast three HLA class I of patient XYZ (Table 38). In addition, eachpeptide is optimized to bind the maximum number of HLA class II of thepatient.

TABLE 38 XYZ ovarian cancer patient's personalized vaccine MAX MAXTarget Antigen HLA HLA XYZ's vaccine Antigen Expression 20mer peptidesclassI classII POC01_P1 AKAP4 89% NSLQKQLQAVLQWIAASQFN 3 5 POC01_P2BORIS 82% SGDERSDEIVLTVSNSNVEE 4 2 POC01_P3 SPAG9 76%VQKEDGRVQAFGWSLPQKYK 3 3 POC01_P4 OY-TES-1 75% EVESTPMIMENIQELIRSAQ 3 4POC01_P5 SP17 69% AYFESLLEKREKTNFDPAEW 3 1 POC01_P6 WT1 63%PSQASSGQARMFPNAPYLPS 4 1 POC01_P7 HIWI 63% RRSIAGEVASINEGMTRWFS 3 4POC01_P8 PRAME 60% MQDIKMILKMVQLDSIEDLE 3 4 POC01_P9 AKAP-3 58%ANSVVSDMMVSIMKTLKIQV 3 4 POC01_P10 MAGE-A4 37% REALSNKVDELAHFLLRKYR 3 2POC01_P11 MAGE-A9 37% ETSYEKVINYLVMLNAREPI 3 4 POC01_P12a MAGE-A10 52%DVKEVDPTGHSFVLVTSLGL 3 4 POC01_P12b BAGE 30% SAQLLQARLMKEESPVVSWR 3 2

Eleven PEPI3 peptides in this immunotherapy composition can induce Tcell responses in XYZ with 84% probability and the two PEPI4 peptides(P0001-P2 and P0001-P5) with 98% probability, according to thevalidation of the PEPI Test shown in Table 3. T cell responses target 13antigens expressed in ovarian cancers. Expression of these cancerantigens in patient XYZ was not tested. Instead the probability ofsuccessful killing of cancer cells was determined based on theprobability of antigen expression in the patient's cancer cells and thepositive predictive value of the ≥1 PEPI3+ Test (AGP count). AGP countpredicts the effectiveness of a vaccine in a subject: Number of vaccineantigens expressed in the patient's tumor (ovarian adenocarcinoma) withPEPI. The AGP count indicates the number of tumor antigens that vaccinerecognizes and induces a T cell response against the patient's tumor(hit the target). The AGP count depends on the vaccine-antigenexpression rate in the subject's tumor and the HLA genotype of thesubject. The correct value is between 0 (no PEPI presented by expressedantigen) and maximum number of antigens (all antigens are expressed andpresent a PEPI).

The probability that patient XYZ will express one or more of the 12antigens is shown in FIGS. 22A-B. AGP95=5, AGP50=7.9, mAGP=100%, AP=13.

A pharmaceutical composition for patient XYZ may be comprised of atleast 2 from the 13 peptides (Table 38), because the presence in avaccine or immunotherapy composition of at least two polypeptidefragments (epitopes) that can bind to at least three HLA of anindividual (≥2 PEPI3+) was determined to be predictive for a clinicalresponse. The peptides are synthetized, solved in a pharmaceuticallyacceptable solvent and mixed with an adjuvant prior to injection. It isdesirable for the patient to receive personalized immunotherapy with atleast two peptide vaccines, but preferable more to increase theprobability of killing cancer cells and decrease the chance of relapse.

For treatment of patient XYZ the 12 peptides were formulated as 4×¾peptide (P0001/1, P0001/2, P0001/3, P0001/4). One treatment cycle isdefined as administration of all 13 peptides within 30 days.

Patient History:

Diagnosis: Metastatic ovarian adenocarcinoma

Age: 51

Family anamnesis: colon and ovary cancer (mother) breast cancer(grandmother)

Tumor Pathology:

BRCA1-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCH1-S2450N

-   -   2011: first diagnosis of ovarian adenocarcinoma; Wertheim        operation and chemotherapy; lymph node removal    -   2015: metastasis in pericardial adipose tissue, excised    -   2016: hepatic metastases    -   2017: retroperitoneal and mesenteric lymph nodes have        progressed; incipient peritoneal carcinosis with small        accompanying ascites        Prior Therapy:    -   2012: Paclitaxel-carboplatin (6×)    -   2014: Caelyx-carboplatin (lx)    -   2016-2017 (9 months): Lymparza (Olaparib) 2×400 mg/day, oral    -   2017: Hycamtin inf. 5×2.5 mg (3× one seria/month)

PIT vaccine treatment began on 21 Apr. 2017.

TABLE 39 Patient XYZ peptide treatment schedule Vaccinations Lot #1^(st) cycle 2^(nd) cycle 3^(rd) cycle 4^(th) cycle POC01/1 N1727 21Apr. 2017 16 Jun. 2017 30 Aug. 2017 19 Oct. 2017 POC01/2 N1728 28 Apr.2017 31 May 2017 POC01/3 N1732 16 Jun. 2017 2 Aug. 2017 20 Sep. 2017POC01/4 N1736 15 May 2017 6 Jul. 2017Patient' Tumor MRI Findings (Baseline Apr. 15, 2016)

-   -   Disease was confined primarily to liver and lymph nodes. The use        of MRI limits detection of lung (pulmonary) metastasis    -   May 2016-January 2017: Olaparib treatment    -   December/25/2016 (before PIT vaccine treatment) There was        dramatic reduction in tumor burden with confirmation of response        obtained at FU2    -   January-March 2017—TOPO protocol (topoisomerase)    -   April/6/2017 FU3 demonstrated regrowth of existing lesions and        appearance of new lesions leading to disease progression    -   Apr. 21, 2017 START PIT    -   July/21/17 (after the 2^(nd) Cycle of PIT) FU4 demonstrated        continued growth in lesions and general enlargement of pancreas        and abnormal para pancreatic signal along with increased ascites    -   July/26/17—CBP+Gem+Avastin    -   September/20/17 (after 3 Cycles of PIT) FU5 demonstrated        reversal of lesion growth and improved pancreatic/parapancreatic        signal. The findings suggest pseudo progression    -   November 28/17 (after 4 Cycles of PIT) FU6 demonstrated best        response with resolution of non-target lesions        MRI data for patient XYZ is shown in Table 40 and FIG. 23 .

TABLE 40 Summary Table of Lesions Responses Lesion/ Baseline FU1 FU2 FU3FU4 FU5 FU6 Best PD Time (%Δ (%Δ (%Δ (%Δ (%Δ (%Δ (%Δ Response Time Pointfrom BL) from BL) from BL) from BL) from BL) from BL) from BL) CyclePoint TL1 NA −56.1 −44.4 −44.8 +109.3 −47.8 −67.3 FU6 FU4 TL2 NA −100.0−100.0 −47.1 −13.1 −100.0 −100.0 FU1 FU3 TL3 NA −59.4 −62.3 −62.0 −30.9−66.7 −75.9 FU6 FU4 TL4 NA −65.8 −100.0 −100.0 −100.0 −100.0 −100.0 FU2NA SUM NA −66.3 −76.0 −68.9 −23.5 −78.2 −85.2 FU6 FU4

Example 22 Design of Personalised Immunotherapy Composition forTreatment of Breast Cancer

The HLA class I and class II genotype of metastatic breast cancerpatient ABC was determined from a saliva sample. To make a personalizedpharmaceutical composition for patient ABC twelve peptides wereselected, each of which met the following two criteria: (i) derived froman antigen that is expressed in breast cancers, as reported in peerreviewed scientific publications; and (ii) comprises a fragment that isa T cell epitope capable of binding to at least three HLA class I ofpatient ABC (Table 41). In addition, each peptide is optimized to bindthe maximum number of HLA class II of the patient. The twelve peptidestarget twelve breast cancer antigens. The probability that patient ABCwill express one or more of the 12 antigens is shown in FIG. 24 .

TABLE 41 12 peptides for ABC breast cancer patient BRC09 vaccine TargetAntigen MAXHLA MAXHLA peptides Antigen Expression 20mer peptide Class IClass II PBRC01_cP1 FSIP1 49% ISDTKDYFMSKTLGIGRLKR 3 6 PBRC01_cP2 SPAG988% FDRNTESLFEELSSAGSGLI 3 2 PBRC01_cP3 AKAP4 85% SQKMDMSNIVLMLIQKLLNE 36 PBRC01_cP4 BORIS 71% SAVFHERYALIQHQKTHKNE 3 6 PBRC01_cP5 MAGE-A11 59%DVKEVDPTSHSYVLVTSLNL 3 4 PBRC01_cP6 NY-SAR-35 49% ENAHGQSLEEDSALEALLNF 32 PBRC01_cP7 HOM-TES-85 47% MASFRKLTLSEKVPPNHPSR 3 5 PBRC01_cP8 NY-BR-147% KRASQYSGQLKVLIAENTML 3 6 PBRC01_cP9 MAGE-A9 44% VDPAQLEFMFQEALKLKVAE3 8 PBRC01_cP10 SCP-1 38% EYEREETRQVYMDLNNNIEK 3 3 PBRC01_cP11 MAGE-A137% PEIFGKASESLQLVFGIDVK 3 3 PBRC01_cP12 MAGE-C2 21%DSESSFTYTLDEKVAELVEF 4 2

Predicted efficacy: AGP95=4; 95% likelihood that the PIT Vaccine inducesCTL responses against 4 CTAs expressed in the breast cancer cells ofBRC09. Additional efficacy parameters: AGP50=6.3, mAGP=100%, AP=12.

Detected efficacy after the 1^(st) vaccination with all 12 peptides: 83%reduction of tumor metabolic activity (PET CT data).

For treatment of patient ABC the 12 peptides were formulated as 4×3peptide (PBR01/1, PBR01/2, PBR01/3, PBR01/4). One treatment cycle isdefined as administration of all 12 different peptide vaccines within 30days.

Patient History

Diagnosis: bilateral metastatic breast carcinoma: Right breast is ERpositive, PR negative, Her2 negative; Left Breast is ER, PR and Her2negative.

First diagnosis: 2013 (4 years before PIT vaccine treatment)

2016: extensive metastatic disease with nodal involvement both above andbelow the diaphragm Multiple liver and pulmonar metastases.

2016-2017 treatment: Etrozole, Ibrance (Palbocyclib) and Zoladex

Results

Mar. 7, 2017: Prior PIT Vaccine treatment

Hepatic multi-metastatic disease with truly extrinsic compression of theorigin of the choledochal duct and massive dilatation of the entireintrahepatic biliary tract. Celiac, hepatic hilar and retroperitonealadenopathy

May 26, 2017: After 1 cycle of PIT

Detected efficacy: 83% reduction of tumor metabolic activity (PET CT)liver, lung lymphnodes and other metastases.

Detected safety: Skin reactions

Local inflammation at the site of the injections within 48 hoursfollowing vaccine administrations

Follow Up:

BRC-09 was treated with 5 cycles of PIT vaccine. She was feeling verywell and she refused a PET CT examination in September 2017. In Novembershe had symptoms, PET CT scan showed progressive disease, but sherefused all treatments. In addition, her oncologist found out that shedid not take Palbocyclib since spring/summer. Patient ABC passed away inJanuary 2018.

The combination of pablocyclib and the personalised vaccine was likelyto have been responsible for the remarkable early response observedfollowing administration of the vaccine. Palbocyclib has been shown toimprove the activity of immunotherapies by increases CTA presentation byHLAs and decreasing the proliferation of Tregs: (Goel et al. Nature.2017:471-475). The PIT vaccine may be used as add-on to the state-of-arttherapy to obtain maximal efficacy.

Example 23— Personalised Immunotherapy Composition for Treatment OfPatient with Late Stage Metastatic Breast Cancer

Patient BRCO5 was diagnosed with inflammatory breast cancer on the rightwith extensive lymphangiosis carcinomatose. Inflammatory breast cancer(IBC) is a rare, but aggressive form of locally advanced breast cancer.It's called inflammatory breast cancer because its main symptoms areswelling and redness (the breast often looks inflamed). Mostinflammatory breast cancers are invasive ductal carcinomas (begin in themilk ducts). This type of breast cancer is associated with theexpression of oncoproteins of high risk Human Papilloma Virus. Indeed,HPV16 DNA was diagnosed in the tumor of this patient.

Patient's stage in 2011 (6 years prior to PIT vaccine treatment): T4:Tumor of any size with direct extension to the chest wall and/or to theskin (ulceration or skin nodules) pN3a: Metastases in ≥10 axillary lymphnodes (at least I tumor deposit >2.0 mm); or metastases to theinfraclavicular (level III axillary lymph) nodes.

14 vaccine peptides were designed and prepared for patient BRCO5 (Table42). Peptides PBRC05-P01-P10 were made for this patient based onpopulation expression data. The last 3 peptides in the Table 42 (SSX-2,MORC, MAGE-B1) were designed from antigens that expression was measureddirectly in the tumor of the patient.

TABLE 42 Vaccine peptides for patient BRC05 BRC05 vaccine Target AntigenMAXHLA MAXHLA peptides Antigen Expression 20mer peptide Class I Class IIPBRC05_P1 SPAG9 88% XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P2 AKAP4 85%XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P3 MAGE-A11 59% XXXXXXXXXXXXXXXXXXXX 3 3PBRC05_P4 NY-SAR-35 49% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P5 FSIP1 49%XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P6 NY-BR-1 47% XXXXXXXXXXXXXXXXXXXX 3 4PBRC05_P7 MAGE-A9 44% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P8 SCP-1 38%XXXXXXXXXXXXXXXXXXXX 3 6 PBRC05_P9 MAGE-A1 37% XXXXXXXXXXXXXXXXXXXX 3 3PBRC05_P10 MAGE-C2 21% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P11 MAGE-A12 13%XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P12 SSX-2 6% XXXXXXXXXXXXXXXXXXXX 3 1PBRC05_P13 MORC ND XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P14 MAGE-B1 NDXXXXXXXXXXXXXXXXXXXX 3 3

T cell responses were measured cells in peripheral mononuclear cells 2weeks after the 1^(st) vaccination with the mix of peptides PBRC05_P1,PBRC05_P2, PBRC05_P3, PBRC05_P4, PBRC05_P5, PBRC05_P6, PBRC05_P7.

T cell responses were measured cells in peripheral mononuclear cells 2weeks after the 1^(st) vaccination with the mix of peptides PBRCO5_P1,PBRCO5_P2, PBRC05_P3, PBRCO5_P4, PBRC05_P5, PBRC05_P6, PBRC05_P7.

TABLE 43 Antigen specific T cell responses: Number of spots/300,000 PBMCAntigen Stimulant Exp1 Exp2 Average SPAG9 PBRC05_P1 2 1 1.5 AKAP4PBRC05_P2 11 4 7.5 MAGE-A11 PBRC05_P3 26 32 29 NY-SAR-35 PBRC05_P4 472497 484.5 FSIP1 PBRC05_P5 317 321 319 NY-BR-1 PBRC05_P6 8 12 10 MAGE-A9PBRC05_P7 23 27 25 None Negative Control (DMSO) 0 3 1.5

The results show that a single immunization with 7 peptides inducedpotent T cell responses against 3 out of the 7 peptides demonstratingpotent MAGE-All, NY-SAR-35, FSIP1 and MAGE-A9 specific T cell responses.There were weak responses against AKAP4 and NY-BR-1 and no responseagainst SPAG9.

REFERENCES

¹ Bagarazzi et al. Immunotherapy against HPV16/18 generates potent TH1and cytotoxic cellular immune responses. Science Translational Medicine.2012; 4(155):155ra138.

² Gudmundsdotter et al. Amplified antigen-specific immune responses inHIV-1 infected individuals in a double blind DNA immunization andtherapy interruption trial. Vaccine. 2011; 29(33):5558-66.

-   ³ Bioley et al. HLA class I—associated immunodominance affects CTL    responsiveness to an ESO recombinant protein tumor antigen vaccine.    Clin Cancer Res. 2009; 15(1):299-306.-   ⁴ Valmori et al. Vaccination with NY-ESO-1 protein and CpG in    Montanide induces integrated antibody/Th1 responses and CD8 T cells    through cross-priming. Proceedings of the National Academy of    Sciences of the United States of America. 2007; 104(21):8947-52.-   ⁵ Yuan et al. Integrated NY-ESO-1 antibody and CD8+ T-cell responses    correlate with clinical benefit in advanced melanoma patients    treated with ipilimumab. Proc Natl Acad Sci USA. 2011; 108(40):    16723-16728.-   ⁶ Kakimi et al. A phase I study of vaccination with NY-ESO-lf    peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients    with cancers expressing the NY-ESO-1 antigen. Int J Cancer. 2011;    129(12):2836-46.-   ⁷ Wada et al. Vaccination with NY-ESO-1 overlapping peptides mixed    with Picibanil OK-432 and montanide ISA-51 in patients with cancers    expressing the NY-ESO-1 antigen. J Immunother. 2014; 37(2): 84-92.-   ⁸ Welters et al. Induction of tumor-specific CD4+ and CD8+ T-cell    immunity in cervical cancer patients by a human papillomavirus type    16 E6 and E7 long peptides vaccine. Clin. Cancer Res. 2008;    14(1):178-87.-   ⁹ Kenter et al. Vaccination against HPV-16 oncoproteins for vulvar    intraepithelial neoplasia. N Engl J Med. 2009; 361(19):1838-47.-   ¹⁰ Welters et al. Success or failure of vaccination for    HPV16-positive vulvar lesions correlates with kinetics and phenotype    of induced T-cell responses. PNAS. 2010; 107(26):11895-9.-   ¹¹ www.ncbi.nlm.nih.gov/projects/gv/mhc/main.fcgi?cmd=init The MHC    database, NCBI (Accessed Mar. 7, 2016).-   ¹² Karkada et al. Therapeutic vaccines and cancer: focus on    DPX-0907. Biologics. 2014; 8:27-38.-   ¹³ Butts et al. Randomized phase IIB trial of BLP25 liposome vaccine    in stage IlIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;    23(27):6674-81.-   ¹⁴ Yuan et al. Safety and immunogenicity of a human and mouse gp100    DNA vaccine in a phase I trial of patients with melanoma. Cancer    Immun. 2009; 9:5.-   ¹⁵ Kovjazin et al. ImMucin: a novel therapeutic vaccine with    promiscuous MHC binding for the treatment of MUC1-expressing tumors.    Vaccine. 2011; 29(29-30):4676-86.-   ¹⁶ Cathcart et al. Amultivalent bcr-abl fusion peptide vaccination    trial in patients with chronic myeloid leukemia. Blood. 2004;    103:1037-1042.-   ¹⁷ Chapuis et al. Transferred WT1-reactive CD8+ T cells can mediate    antileukemic activity and persist in post-transplant patients. Sci    Transl Med. 2013; 5(174):174ra27.-   ¹⁸ Keilholz et al. A clinical and immunologic phase 2 trial of Wilms    tumor gene product 1 (WT1) peptide vaccination in patients with AML    and MDS. Blood; 2009; 113(26):6541-8.-   ¹⁹ Walter et al. Multipeptide immune response to cancer vaccine    IMA901 after single-dose cyclophosphamide associates with longer    patient survival. Nat Med. 2012; 18(8):1254-61.-   ²⁰ Phuphanich et al. Phase I trial of a multi-epitope-pulsed    dendritic cell vaccine for patients with newly diagnosed    glioblastoma. Cancer Immunol Immunother. 2013; 62(1):125-35.-   ²¹ Kantoff et al. Overall survival analysis of a phase II randomized    controlled trial of a Poxviral-based PSA-targeted immunotherapy in    metastatic castration-resistant prostate cancer. J Clin Oncol. 2010;    28(7): 1099-105.-   ²² Tagawa et al. Phase I study of intranodal delivery of a plasmid    DNA vaccine for patients with Stage IV melanoma. Cancer. 2003;    98(1):144-54.-   ²³ Slingluff et al. Randomized multicenter trial of the effects of    melanoma-associated helper peptides and cyclophosphamide on the    immunogenicity of a multipeptide melanoma vaccine. J Clin Oncol.    2011; 29(21):2924-32.-   ²⁴ Kaida et al. Phase 1 trial of Wilms tumor 1 (WT1) peptide vaccine    and gemcitabine combination therapy in patients with advanced    pancreatic or biliary tract cancer. J Immunother. 2011; 34(1):92-9.-   ²⁵ Fenoglio et al. A multi-peptide, dual-adjuvant telomerase vaccine    (GX301) is highly immunogenic in patients with prostate and renal    cancer. Cancer Immunol Immunother; 2013; 62:1041-1052.-   ²⁶ Krug et al. WT1 peptide vaccinations induce CD4 and CD8 T cell    immune responses in patients with mesothelioma and non-small cell    lung cancer. Cancer Immunol Immunother; 2010; 59(10): 1467-79.-   ²⁷ Slingluff et al. Clinical and immunologic results of a randomized    phase II trial of vaccination using four melanoma peptides either    administered in granulocyte-macrophage colony-stimulating factor in    adjuvant or pulsed on dendritic cells. J Clin Oncol; 2003;    21(21):4016-26.-   ²⁸ Hodi et al. Improved survival with ipilimumab in patients with    metastatic melanoma. N Engl J Med; 2010; 363(8):711-23.-   ²⁹ Carmon et al. Phase I/II study exploring ImMucin, a pan-major    histocompatibility complex, anti-MUC1 signal peptide vaccine, in    multiple myeloma patients. Br J Hematol. 2014; 169(1):44-56.-   ³⁰    www.merckgroup.com/en/media/extNewsDetail.html?newsId=EB4A46A2AC4A52E7C1257AD9    001F3186&newsType=1(Accessed Mar. 28, 2016)-   ³¹ Trimble et al. Safety, efficacy, and immunogenicity of VGX-3100,    a therapeutic synthetic DNA vaccine targeting human papillomavirus    16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia    2/3: a randomised, double-blind, placebo-controlled phase 2b trial.    Lancet. 2015; 386(10008):2078-88.-   ³² Cusi et al. Phase I trial of thymidylate synthase poly epitope    peptide (TSPP) vaccine in advanced cancer patients. Cancer Immunol    Immunother; 2015; 64:1159-1173.-   ³³ Asahara et al. Phase I/II clinical trial using HLA-A24-restricted    peptide vaccine derived from KIF20A for patients with advanced    pancreatic cancer. J Transl Med; 2013; 11:291.-   ³⁴ Yoshitake et al. Phase II clinical trial of multiple peptide    vaccination for advanced head and neck cancer patients revealed    induction of immune responses and improved OS. Clin Cancer Res;    2014; 21(2):312-21.-   ³⁵ Okuno et al. Clinical Trial of a 7-Peptide Cocktail Vaccine with    Oral Chemotherapy for Patients with Metastatic Colorectal Cancer.    Anticancer Res; 2014; 34: 3045-305.-   ³⁶ Rapoport et al. Combination Immunotherapy after ASCT for Multiple    Myeloma Using MAGE-A3/Poly-ICLC Immunizations Followed by Adoptive    Transfer of Vaccine-Primed and Costimulated Autologous T Cells. Clin    Cancer Res; 2014; 20(5): 1355-1365.-   ³⁷ Greenfield et al. A phase I dose-escalation clinical trial of a    peptidebased human papillomavirus therapeutic vaccine with Candida    skin test reagent as a novel vaccine adjuvant for treating women    with biopsy-proven cervical intraepithelial neoplasia 2/3.    Oncoimmunol; 2015; 4:10, e1031439.-   ³⁸ Snyder et al. Genetic basis for clinical response to CTLA-4    blockade in melanoma. N Engl J Med. 2014; 371(23):2189-99.-   ³⁹ Van Allen et al. Genomic correlates of response to CTLA-4    blockade in metastatic melanoma. Science; 2015; 350:6257.-   ⁴⁰ Li et al. Thrombocytopenia caused by the development of    antibodies to thrombopoietin. Blood; 2001; 98:3241-3248-   ⁴¹ Takedatsu et al. Determination of Thrombopoietin-Derived Peptides    Recognized by Both Cellular and Humoral Immunities in Healthy Donors    and Patients with Thrombocytopenia. 2005; 23(7): 975-982-   ⁴² Eisenhauer et al. New response evaluation criteria in solid    tumors: revised RECIST guideline (version 1.1). Eur J Cancer; 2009;    45(2):228-47.-   ⁴³ Therasse et al. New guidelines to evaluate the response to    treatment in solid tumors: European Organization for Research and    Treatment of Cancer, National Cancer Institute of the United States,    National Cancer Institute of Canada. J Natl Cancer Inst; 2000;    92:205-216.-   ⁴⁴ Tsuchida & Therasse. Response evaluation criteria in solid tumors    (RECIST): New guidelines. Med Pediatr Oncol. 2001; 37:1-3.-   ⁴⁵ Dune et al. International uniform response criteria for multiple    myeloma. Leukemia; 2006; 20:1467-1473.

What is claimed is:
 1. A method of treating breast cancer usingimmunotherapy in an individual in need thereof, the method comprising:administering to the individual a pharmaceutical composition comprising(i) a polypeptide comprising at least a first peptide and a secondpeptide, wherein each of the first and the second peptide is selectedfrom a peptide comprising the amino acid sequence of any of SEQ ID NOs:41-60, 64, or 195-233; and (ii) a first pharmaceutically acceptableadjuvant.
 2. The method of claim 1, wherein each of the at least thefirst peptide and the second peptide each comprises the amino acidsequence of any of SEQ ID NOs: 81-111 or 435-449.
 3. The method of claim1, wherein the composition further comprises at least one additionalpolypeptide comprising at least one peptide comprising a fragment of anantigen selected from TSGA10, PIWIL-2, AKAP4, PLU-1, RHOXF-2, SPAG9,EpCam, HIWI, ODF-4, NY-SAR-35, RHOXF-2, PRAIVIE, MAGE-A9, MAGE-A11,SP17, BORIS, Survivin, HOM-TES-95, NY-BR-1, and NY-ESO-1.
 4. The methodof claim 1, wherein the composition further comprises at least oneadditional polypeptide comprising at least one peptide comprisingfragment of an antigen selected from AKAP4, BORIS, SPAG9, NY-SAR-35,PRAME, SURVIVIN, MAGE-A9, MAGE-A11, NY-BR-1 and HomTes85.
 5. The methodof claim 1, wherein the composition further comprises at least oneadditional pharmaceutically acceptable adjuvant, diluent, carrier,preservative, excipient, buffer, stabilizer, or combination thereof. 6.The method of claim 1, wherein the composition further comprises atleast one additional pharmaceutically acceptable adjuvant.
 7. The methodof claim 1, wherein the pharmaceutically acceptable adjuvant is selectedfrom the group consisting of water-in-oil emulsions, QS-21, GM-CSF,cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum,levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB),keyhole limpet hemocyanins (KLH), Freunds complete adjuvant, Freundsincomplete adjuvant, mineral gels, aluminum hydroxide (Alum),lysolecithin, pluronic polyols, polyanions, oil emulsions,dinitrophenol, diphtheria toxin (DT), and combinations thereof.
 8. Themethod of claim 1, further comprising administering a chemotherapeuticagent, a checkpoint inhibitor, a targeted therapy, radiation therapy,another immunotherapy, or combination thereof to the individual.
 9. Themethod of claim 5, wherein the at least one additional pharmaceuticallyacceptable adjuvant is selected from the group consisting ofwater-in-oil emulsions, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds complete adjuvant, Freunds incomplete adjuvant, mineralgels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.
 10. The method of claim 6, wherein the at leastone additional pharmaceutically acceptable adjuvant is selected from thegroup consisting of water-in-oil emulsions, QS-21, GM-CSF,cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum,levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB),keyhole limpet hemocyanins (KLH), Freunds complete adjuvant, Freundsincomplete adjuvant, mineral gels, aluminum hydroxide (Alum),lysolecithin, pluronic polyols, polyanions, oil emulsions,dinitrophenol, diphtheria toxin (DT), and combinations thereof.